Consulted with our advisor, Frank, to advise researchers and scientists
Reached out to scientists at Mt Sinai Hospital, Jason Fuller and Russell Hanson
Met with Jason Fuller and Russell Hanson, scientists at Genetics Department at Mt Sinai Hospital
Scheduled weekly meetings for the rest of the year
Week 2 (March 26-Apr 1)
Brainstormed for a potential topic (Sickle Cell Anemia, Cystic Fibrosis, Huntington’s Disease); decided on HD, proposed by Yeji Cho
Determined project leaders of the project
Divided into subsections (website, design, fundraising, social media, computational) and assigned roles, such as other team leads
Week 3 (Apr 2-8)
Determined possible method to target the disease
Outlined the abstract for the project
Week 4 (Apr 9-15)
Took headshots of members and team pictures to put on website
Administered Drug Tests for medical clearance
HIPAA (Health Insurance Portability and Accountability Act) Training
Fire Drill Practice
Week 5 (Apr 16-22)
Decided to use WordPress as website template
Happily received our lab coats and Mount Sinai IDs
Week 6 (Apr 23-29)
Submitted payment for iGEM group registration
Signed consent forms
Week 7 (Apr 30-May 6)
Designed graphic of brain and neuron for website
Week 8 (May 7-13)
Brainstormed for a team name and designed logo; decided on HD Resolution
Week 9 (May 14-20)
Designed T-shirts, hats, and sweaters
Brainstormed for companies/sponsors to partner with us
Received iGEM parts kit
Week 10 (May 21-27)
Organized home page of website
Created sponsorship/partnership letters for possible companies
Week 11 (May 28-Jun 3)
Julia, Erik and Stacy organized a table at Peace Angels Benefit Concert at Steinway and Sons Concert Hall in May 28, 2017
Mailed out consent forms
Scheduled wetlab training dates for the summer
Week 12 (Jun 4-10)
Early Training: lab safety, project overview, practiced lab techniques such as pipetting, centrifugation, PCR, etc.
Week 13 (Jun 11-17)
Organized team page of website
Contacted over 50 private and public companies in pharmaceutical and biotech industries, non-profit organizations for sponsorship and support.
Week 14 (Jun 18-24)
Created Twitter account
Christi and Marianne completed safety form 1, 2, 3
Week 15 (Jun 25-Jul 1)
Jason, Justin, Jeewhan, Marianne met up to analyze the data collection
Week 16 (Jul 2-8)
Computational Lab Weeks:
First weeks we talked about how to get an application called mFold
mFold helps to create a simulation of the RNA bonds fusing instead having to actual do it experimentally
Week 17 (Jul 9-15)
Computational Lab Weeks (continued):
Looked for Huntington’s mRNA in particular in NCBI
NM numbers, other prefixes gave types of mRNA
Found Huntington’s-related sequences in NCBI database (assumed at least 5-10 sequences)
Week 18 (Jul 16-22)
Christi, Rachel, Esther, and Marianne organized a fundraising and information table at Dominico-American Society of Queens in Flushing Town Hall in Flushing, Queens NY in July 22 2017
Created Facebook account
Created GoFundMe page
Week 19 (Jul 23-29)
Received over 100 likes on Facebook page thanks to our Social Media and Outreach team.
Computational Data:
We used data we received from [ncbi.com] and used it to compare with the wild type and the infected types.
Found 2 accession numbers- NM_002111.8 (mRNA) and NP_002102.4 (protein); however, only one was mRNA and none were disease form
We then collected the data in our shared folder
Looked through other databases for other mutants; found:
Study with “20 Huntington’s Disease and 49 neurologically normal control samples from post-mortem human subjects”
Re-calculated sequences with artificially added CAG repeats (added 42 and 60 repeats)
Week 21 (Aug 6-12)
Wrote letters to respective schools to ask for partnerships
Manually analyzed results of ViennaRNA calculations for chaperone/guideRNA hairpin targets.
Determined that this process was less reproducible than desired, and it worried the most viable hairpin loop might be buried in the the tertiary structure
Decided to change approach: Use RNAi tools to calculate the toe-hold footing (areas in RNA that can be used to initiate strand displacement and/or break apart mRNA secondary structures).
Spent remainder of the day testing the following tools:
We used to addgene.com to find plasmids that had less than 26Q repeats.
We used a genome to protein sequence converter called [] in order to compare the wild type plasmid and the infected.
At first we thought we had a good candidate with 20Q that was very similar, however, didn’t work because the sequence didn’t completely match up with the wild type.
Grew cell subcultures of E. Coli for future testing
Familiarized ourselves with autoclaving and basic sterilizing processes
Learned how to store E. coli for future experiments
HDSA agreed to be sponsor
Week 23 (Aug 20-26)
Learned about transfection and tested cells for competency
Updated team Wiki’s page on the iGem website
Reached out to our respective high schools for sponsorship
Hereditary Disease Foundation/Huntington’s Disease Foundation agreed to be sponsor
Week 24 (Aug 27-Sept 2)
Jessica, Erin, Catherine, and Yunsu helped organize a presentation table at Dominico-American Society of Queens Benefit Concert at National Opera America Center in New York NY on August 30 2017
Reached out to Cerebral Palsy Alliance Research Foundation about partnering and sponsorship and discussed creating a joint campaign CPARF’s Steptember campaign to promote 10,000 steps a day goal
Registered team members and created a team roster
Testing Competency of Cells
Project: HD Resolution iGEM Notebook
Authors: HD Resolution iGEM
Date: 2017-09-19
Tuesday, 9/19/17
1.
Clean working area with 10% ethanol
2.
Throw competent cells on ice
3.
Label one 1.5 mL microcentrifuge tubes for each transformation and pre-chill on ice.
a.
Orange = 50 pg/μl
b.
Yellow = 10 pg/μl
c.
Red = 100 pg/μl
4.
Spin down DNA tubes from competent cell test kit. Quick spin of 20-30 seconds at 8000-10000rpm
5.
Pipet 10μl of DNA into centrifuge tubes. 50μl of competent cells already aliquoted. Flick gentlly
6.
Incubate on ice for 30 min.
7.
Heatshock cells into water bath of 42 degrees Celsius for 45 sec.
8.
Immediately transfer tubes back into ice and incubate on ice for 5 min
9.
Add 950 μl of SOC Media per tube and incubate at 37 degrees Celsius for 1 hour, shaking at 200-300rpm.
10.
Pipet 100μl from each tube onto appropriate plate, spread mixture evenly.
11.
Incubate at 37 degrees Celsius overnight/16 hrs. Place plates with agar side on top and lid on bottom.
Untitled
Project: HD Resolution iGEM Notebook
Authors: HD Resolution iGEM
Date: 2017-09-20
Wednesday, 9/20/17
1.
Innoculated 600 mL LB w/ 1 mL O/N culture
2.
Shake at 37 degrees Celsius, DDC 600 = 0.4, 3 hr
3.
Cooled all materials in ice, including solutions (CaCl3), cells, tubes, & tips
4.
Aliquoted 600 mL of cells into 12 50 mL tubes, 30 mL each
5.
Spin 1.5 k rcf, 5 min
6.
Decant sup, resus, pellets in 1 mL CaCl2 (cold) by gentle rocking, left on ice for 30 min
7.
Spin as above
8.
Discard supernatent
9.
Added fresh cold 100 mM CaCl3
10.
Resuspend as above
11.
Added 10.2 mL 50% glycerol to 50 mL falcon tube (chilled)
12.
Consolidated all resuspended cells into the tube containing 50% glycerol
13.
Did not fully resuspend cells, still looked chunky
14.
Froze in 1 mL aliquots of liiquid nitrogen, left in unmarked box below iGEM box in -80 degrees Celsius freezer
Continuation
Project: HD Resolution iGEM Notebook
Authors: HD Resolution iGEM
Date: 2017-09-22
Friday, 9/22/17
1.
Added 175μl of ice cold Alkaline Lysis Solution III through the viscous bacterial lysate. Inverted tube several times and stored tube on ice for 5 min.
2.
Centrifuged lysate at maximum speed for 5 min at 4 degrees Celsius, and transferrred the supernatant to a new tube
3.
Precipitated the nucleic acids from the supernatant. Added 1 mL of ethanol at room temperature. Mixedsolution by vortexing and allowed mixture to stand for two minutes at room temp.
4.
Collected precipitated nucleic acds by centrifugation at max speed for 5 min at 4 degrees Celsius. Dropped precipitated nucleic aicds. Redid centrifugation at max spped for 5 minutes at 4 degrees Celsius.
5.
Removed supernatant by gentle aspiration, as shown in step 4. Stood tube in an inverted position on a paper towel to allow all the fluid to drain away. Used a kimwipe to remove the drops of fluid adhering to the walls of the tube.
6.
Added 1 mL of 70% ethanol to the pellet and inverted the closed tube several times.
7.
Recovered DNA by centrifugation at max speed for 2 min at 4 degrees Celsius in a microcentrifuge.
Transformation
Project: HD Resolution iGEM Notebook
Authors: HD Resolution iGEM
Date: 2017-09-25
Monday, 9/25/17
1.
Thaw DHS-Alpa Cells on ice
2.
Add 2 mL of DNA to cells (from previously isolated adgene plasmids)
3.
Stand for 34 min on ice
4.
Cells are incubated for 60 sec at 42 degrees Celsius
5.
Put cells on ice for 5 minut s
6.
Add 900μl SOC media
Preparation of LB-Agar Plates
Project: HD Resolution iGEM Notebook
Authors: HD Resolution iGEM
Date: 2017-09-26
Tuesday, 9/26/17
Preparation of LB-Agar Plates
- Adapted from CSH Protocols
LB Agar
- 3.75 gramf of Agar
- 250 mL of 1x LB Medium
1.
Mix
2.
Autoclave on Liquid Cycle for 30 minutes
a.
Do not overfill bottles
I.
250 mL of LB was autoclaved in a 500 mL bottle to prevent it from boiling over.
3.
Add 2.5 mL of Kanamycin Stock (25 mg/mL) to a final concentration of 25ug/mL, once LB-Agar has coded to about 50 degrees Celsius.
4.
Heat on a hot plate to re-melt sugar
Running Electrophoresis Gel of ADDGENE Plasmid Digests
Project: HD Resolution iGEM Notebook
Authors: HD Resolution iGEM
Date: 2017-09-26
Tuesday, 9/26/17
1.
Cut up unused gel and add to remaining unused 2% agarose state.
Spin down DNA from competent cell test kit and interlab DNA
5.
Pipet 1μl of DN from competent cell test kit into tubes labeled 1, 4, 5, 6, neg
6.
Pipet 85μl of competent cells into all tubes, flick gently to mix
7.
Incubate on ice for 30 minutes
8.
Heat shock cells by placing tubes into a hot water bath at 42 degrees Celsius for 45 seconds
9.
Transfer back and incubate on ice for 5 min
10.
Add 45μl of SOC Media into eah cand incubate at 37 degrees for 1 hour shaking at 200-300 rpm
11.
Warm up agar plates, label
12.
Pipet 100μl from each tube onto appropriate plate, spread mixture evenly across plates via sterile glass beads.
13.
Close with parafilm
14.
Incubate at 37 degrees Celsius overnight w/ plates positioned w/ agar side at top and lid at bottom
Interlab
Project: HD Resolution iGEM Notebook
Authors: HD Resolution iGEM
Date: 2017-09-27
Wednesday, 9/27/17
1.
Clean working area
2.
Pipette 1μl of 1, 4, 5, 6, neg - and control each with their respective cell tube
3.
INcubate for 30 min (until 7:02)
4.
Pipette 100μl of LUDOX into wells A1, B1, C1. D1 each
5.
Pipette 100μl of H2O each into wells A2, B2, C2, D2
6.
Spin down fluorescein tube
7.
Resuspend 100μM florescein solution in 1 mL 1xPBS to make 2x solution
8.
Dilute to 1x.
9.
Perform serial dilution with 10 columns of 1x PBS (leave an additional column as regular PBS)
Restriction Digest on Previously Isolated DNA Plasmid
Project: HD Resolution iGEM Notebook
Authors: HD Resolution iGEM
Date: 2017-10-06
Friday, 10/6/17
1.
Placed previously isolated DNA plasmid on ie (61 and 62)
2.
Measured the concentration of the DNA Plasmids with the nanodrop (2μl)
a.
DNA plasmid 40261 : 356.2 ng/μl
b.
DNA plasmid 40262 : 300.5 ng/μl
3.
Diluted DNA plasmid 1 : 3 to approximate 100 ng/μl and measured with nanodrop
a.
DNA plasmid 40261 : 117.9 ng/μl
b.
DNA plasmid 40262 : 106.1 ng/μl
4.
Labeled fresh 0.5 mL tubes 61 and 62
5.
For each tube, transferred 5 ul of 10x NEBuffer, 1 ul of Stul, 1 mL of cac 81, and 33 ul of dH2O
6.
Transferred 10μl DNA Plasmid 40261 to 0.5 mL tube 61 and 10μl of DNA plasmid 40262 to 0.5 mL tube 62
7.
Incubated 0.5 mL tubes at 37 degrees Celsius for fifteen minutes
8.
Added 10μl purple 6x loading dye to each of the 0.5 mL tube and mixed
9.
Gel was loaded as follows:
A
B
C
D
E
F
G
1
Cane
1
2
3
4
5
6
2
10μl of 2 log ladder
10μl of digested ladder
10μl of digested 40262
15μl of digested 40262
15μl of digested 40261
15μl of digested 40261
10μl of 2 log ladder
Table1
Re-Running Gel Electrophoresis
Project: HD Resolution iGEM Notebook
Authors: HD Resolution iGEM
Date: 2017-10-10
Tuesday, 10/10/17
1.
Repeat of 10/6/17 procedure from Step 5
2.
Placed unused gel in gel box for 30 min
3.
Pipetted 10μl in 1st and last lane (purple ladder)
4.
Pipetted 15μl in 2nd and 2nd to last lane (3x concentration ol-digest)
5.
Pipetted 30μl (double bond) in outer lanes
6.
Make ETBR solution and place gel in there and on SHAKER (5 min)
7.
Image gel and results: Showed that plasmids were restricted at least once, but 100 bp fragmentts were not seen
8.
Conclusion: Restriction enzymes are to be checked for cut/not.
Re-Run Restrictions on 3x oncnerated samples for 1 hour
Project: HD Resolution iGEM Notebook
Authors: HD Resolution iGEM
Date: 2017-10-12
Thursday, 10/12/17
1.
Labeled 0.5 mL tubes "61" and "62"
2.
Transferred 5μl of 10x NEBuffer, 1μl of StuI, 1μl of Cac 81, and 33μl H2O
3.
Transferred 10μl of DNA plasmid 40261 to 0.5 mL tube "62"
4.
Incubated tubes at 37 degrees Celsius for 1 hour
5.
Added 10μl 6x purple loading dye to each tube and mixed
6.
Gel was labeled as follows
A
B
C
D
E
F
G
H
1
Lane
1
2
3
4
5
6
7
2
empty
10μl 2 log buffer
15μl digested 40261
26μl digested 40261
26μl digested 40262
15μl digested 40262
10μl 2 log buffer
3
Table1
Test of DH5Alfa Souls
Project: HD Resolution iGEM Notebook
Authors: HD Resolution iGEM
Date: 2017-10-12
Thursday, 10/12/17
Gel Extraction and PCR Purification
Project: HD Resolution iGEM Notebook
Authors: HD Resolution iGEM
Date: 2017-10-13
Friday, 10/13/17
1.
Weighed gel slices containing DNA fragment
a.
61a)0.3620g b)0.3895g
b.
62a) 0.2865g b)0.3877g
2.
Added gel solubilization buffer L3 in 3:1 ratio to gel in a 1.7 mL polypropylene tube
3.
Incubated tubes at 50 degrees Celsius for 15 min and inverted tubes every 3 min
4.
Incubated the tubes for an additional 5 min when gel slice appeared dissolved
5.
Pipetted and dissolved gel piece onto a column at 12000 x g for 1 minute. Discarded flow-through and placed column into wash tube
6.
Added 500μl wash buffer containing ethanol to the column. Centrifuged column at >12000 x g for one minute. DIscarded flow thorugh and placed column into wash tube. Centrifuged column at max speed for 2-3 min. Discarded flow-through.
7.
Placed column into recovery tube. Added 50 mL elution buffer (E1) to the column. INcubated tube for a minute at room temperature. Centrifuged tube > 12000 x g/minute.
8.
Stored, purified DNA at -20 degrees Celsius for immediate use
Purifying Gel Bonds from STUL-XBAL Digest
Project: HD Resolution iGEM Notebook
Authors: HD Resolution iGEM
Date: 2017-10-14
Saturday, 10/14/17
1.
weigh gel slices containing dna fragments
a.
61 > 0.2100 g
b.
62 > 0.2230 g
2.
Add Gel solubilization buffer L3 to excised gels in the tubes in a 3:1 ratop
a.
61 > 0. 63 g = 630μl
b.
62 > 0.669 g = 669μl
3.
Incubate at 50 degrees Celsius for 10 minutes ad invert tube every 3 minutes. After the gel slie appears dissolves, incubate tube for an additional 3 min
4.
Add 1 gel volume of isporopanol to dissolved gel slices. Mix well
a.
61 > 210μl
b.
62 > 223μl
5.
Pipet 850μl of dissolved gel pieces into wash tubes. Centrifuge for
How to Assemble the Parts we currently have
Project: HD Resolution Shared Project
Authors: jason fuller
Date: 2017-10-18
Wednesday, 10/18/17
Assuming Exon 1 Flanks have been properly assembled, the remaing ligations could proceed as follows:
Pair and join every 2 G-Blocks, then PCR amplify (IDT doesn't reccomend PCR amplifying parts greater than 1000 bases)
- Start working backwards (GB15 + GB14, GB13+GB12, etc...)
- Pair the Flanked Exon1 part with GB1
Should work as you are only requring polymerase to amplify 500kb or less
OR
LOW EFFICIENCY - extra work, but probably will work!
Pair 3 sets of 5 G-Blocks using mastermix
Blunt cut a plasmid with a restriction enzyme (peferably one with other restriction sites near by - or where a restriction site will be made where the insert is) CUT in the middle of the GFP!!!!!!!
Blunt assemble
transform
colony pick many colonies (THAT DON'T GLOW)
miniprep separtate colonies
- run gel to look for plasmid of correct size. (on side)
Run Gibson assembly.....
OR
remove phosphates from above plasmid cuts via pcr amplification
add phosphates to gibson assembled g-blocks* (how? / how to separate from other smaller fragments?)
Gibson assemble the 8 parts into 2 separate plasmids (could try 1 but success seesm unlikely):
- prepare a unphosphorlated vector backbone (via reverse amplification PCR is best)
- after mastermix step, phosphoralate the DNA
- Vector insert as shown here: https://youtu.be/-Vh-LbJAJpk?t=19m52s
Need to order?: phosphoralase, dephosphoralase (or primers for the backbone), blunt-end lligase, ligase
Questions to answer ASAP:
Do we have Exon1 properly assmebled?
Do we have the primers needed so we don't have to dephosphoralate?
ToeHold Exchange Questions
Project: HD Resolution Shared Project
Authors: jason fuller
Date: 2017-10-18
Wednesday, 10/18/17
Do we need to assemble better Kozak sequences for our syntheitc mRNA replacement?
WT-Mutated = exact gene
chaperone target footing = fagments of exact gene starting at posion 7 up though position 47
- seems like we shouldn't start later than 30 or sooner than 15
- 22 to
image.png
Kozak Sequences should be evaluated and/or Alternative codon used from positions 7 though 47
Chaperone extension though the start codon could therotically go up though the BbCI cut site at position 209 but is perhaps better to keep short and extend no further than the StuI cut site at positon 11O
image.png
Kozak Sequence is imperfect in WT HTT, syntetic form should be changed to contain the GCCG portion:
image.png
Everything before this sequence doesn't really matter and could use alternative splicing.
Perhaps the sequence can just be trimed before this and the guid strand be the hairpin target overhang?
Else it might be nice for it to be D
Wild Type Huntinton mRNA - GBlock Aligned
What did our PCR actually amplify?
Project: HD Resolution Shared Project
Authors: jason fuller
Date: 2017-10-18
Wednesday, 10/18/17
According to PCR theroy the "short" amplicons swamp out all others during an aplification:
http://www.cybertory.org/exercises/primerDesign/#Background (about 1/2 way down)
Thus, we don't actually need perfect cut sites if we are getting enough sticky ends to start an amplification....
From:
Wild Type Huntinton mRNA - GBlock Aligned
image.png
image.png
NEBuilder HiFi DNA Assembly Reaction Protocol
Project: HD Resolution Shared Project
Authors: Sohee
Date: 2017-10-20
Friday, 10/20/17
Optimal Quantities
NEB recommends a total of 0.03–0.2 pmols of DNA fragments when 1 or 2 fragments are being assembled into a vector, and 0.2–0.5 pmols of DNA fragments when 4–6 fragments are being assembled. Efficiency of assembly decreases as the number or length of fragments increases. To calculate the number of pmols of each fragment for optimal assembly, based on fragment length and weight, we recommend the following formula, or using the tool, NEBiocalculator.
pmols = (weight in ng) x 1,000 / (base pairs x 650 daltons)
50 ng of 5000 bp dsDNA is about 0.015 pmols
50 ng of 500 bp dsDNA is about 0.15 pmols
The mass of each fragment can be measured using the NanoDrop instrument, absorbance at 260 nm or estimated from agarose gel electrophoresis followed by ethidium bromide staining.
HiFi DNA Assembly Protocol
1.
Set up the following reaction on ice:
Table 1: Recommended Amount of Fragments Used for Assembly
A
B
C
D
1
2–3 Fragment Assembly*
4–6 Fragment Assembly**
Positive Control✝
2
Recommended DNA Molar Ratio
vector:insert = 1:2
vector:insert = 1:1
3
Total Amount of Fragments
0.03–0.2 pmols*X μl
0.2–0.5 pmols**X μl
10 μl
4
NEBuilderHiFi DNA Assembly Master Mix
10 μl
10 μl
10 μl
5
Deionized H2O
10-X μl
10-X μl
0
6
Total Volume
20 μl✝✝
20 μl✝✝
20 μl
Table2
* Optimized cloning efficiency is 50–100 ng of vector with 2-fold excess of inserts.
Use 5 times more insert if size is less than 200 bp. Total volume of unpurified PCR fragments in the assembly reaction should not exceed 20%.
** To achieve optimal assembly efficiency, design ≥ 20 bp overlap regions between each fragment with equimolarity (suggested: 0.05 pmol each).
† Control reagents are provided for 5 experiments.
†† If greater numbers of fragments are assembled, increase the volume of the reaction, and use additional NEBuilder HiFi DNA Assembly Master Mix.
2. Incubate samples in a thermocycler at 50°C for 15 minutes (when 2 or 3 fragments are being assembled) or 60 minutes (when 4–6 fragments are being assembled). Following incubation, store samples on ice or at –20°C for subsequent transformation.
Note: Extended incubation up to 60 minutes may help to improve assembly efficiency in some cases (for further details see FAQ section).
3. Transform NEB 5-alpha or 10-beta Competent E. coli cells (provided in the cloning kit, bundle or purchased separately from NEB) with 2 μ l of the assembled product, following the transformation protocol.
iGEM Exon 1 Assembly
Project: HD Resolution Shared Project
Authors: Sohee
Date: 2017-10-20
Friday, 10/20/17
HiFi DNA Assembly Protocol
1.
Set up the following reaction on ice:
Table 1: Recommended Amount of Fragments Used for Assembly
A
B
C
D
1
2–3 Fragment Assembly*
4–6 Fragment Assembly**
Positive Control✝
2
Recommended DNA Molar Ratio
vector:insert = 1:2
vector:insert = 1:1
3
Total Number of Fragments***
0.03–0.2 pmols*X μl
0.2–0.5 pmols**X μl
10 μl
4
NEBuilderHiFi DNA Assembly Master Mix
10 μl
10 μl
10 μl
5
Deionized H2O
10-X μl
10-X μl
0
6
Total Volume
20 μl✝✝
20 μl✝✝
20 μl
Table2
* Optimized cloning efficiency is 50–100 ng of vector with 2-fold excess of inserts.
Use 5 times more insert if size is less than 200 bp. Total volume of unpurified PCR fragments in the assembly reaction should not exceed 20%.
** To achieve optimal assembly efficiency, design ≥ 20 bp overlap regions between each fragment with equimolarity (suggested: 0.05 pmol each).
*** When attempting to add flanking reagents, do not add flanking DNA until the reaction mixture has been heated until 50˚C has been reached.
† Control reagents are provided for 5 experiments.
†† If greater numbers of fragments are assembled, increase the volume of the reaction, and use additional NEBuilder HiFi DNA Assembly Master Mix.
2. Incubate samples in a thermocycler at 50°C for 15 minutes (when 2 or 3 fragments are being assembled) or 60 minutes (when 4–6 fragments are being assembled). Following incubation, store samples on ice or at –20°C for subsequent transformation.
Note: Extended incubation up to 60 minutes may help to improve assembly efficiency in some cases (for further details see FAQ section).
3. Transform NEB 5-alpha or 10-beta Competent E. coli cells (provided in the cloning kit, bundle or purchased separately from NEB) with 2 μ l of the assembled product, following the transformation protocol.
G-block Assembly
Project: HD Resolution Shared Project
Authors: HD Resolution iGEM
Date: 2017-10-20
Friday, 10/20/17
Purpose: Begin gibson assembly so that we can PCR once primers are in.
Materials:
IDT G-blocks (15)
NEB Hifi Builder Kit
Nuclease free water
A
B
C
D
1
Concentration Range of DNA fragments
02 - .5 pmols*
.2 - 1.0 pmols*
0 pmols
2
Total Volume of Fragments (μl)
10
3
Gibson Assembly Master Mix (2x) (μl)
10
10
10
4
Deionized Water (μl)
10
10
0
5
Total Volume (μl) ***
20
20
20
Table1
1.
Normalized concentrations of all G-blocks to 10 fmol per microliter.
2.
Take 5 microliters of each G-block sample and put into a new tube of each paired G-block sample.
3.
Then add 10 microliters of Gibson assembly master mix to each tube.
4.
Incubate for 15 minutes at 50 C in the thermocycler.
Gibson Assembly® Protocol (E5510)
Introduction
This is the protocol for Gibson Assembly using the Gibson Assembly® Cloning Kit (E5510). More information from NEB can be found here.
LB (Luria-Bertani) plates with appropriate antibiotic
Procedure
Set up the following reaction on ice:
Reaction volumes: Use this table to calculate reaction volumes and set up the reaction. Remember to input your total DNA fragment volume in cells B3 and C3 for assemblies with 2-3 fragments and 4-6 fragments, respectively.
- NEB recommends a total of 0.02–0.5 pmols of DNA fragments when 1 or 2 fragments are being assembled into a vector and 0.2–1.0 pmoles of DNA fragments when 4–6 fragments are being assembled. Efficiency of assembly decreases as the number or length of fragments increases. To calculate the number of pmols of each fragment for optimal assembly, based on fragment length and weight, we recommend using NEB's online tool, NEBioCalculator.
- The mass of each fragment can be measured using the NanoDrop instrument, absorbance at 260 nm, or estimated from agarose gel electrophoresis followed by ethidium bromide staining.
A
B
C
D
1
2-3 Fragments Assembly
4-6 Fragments Assembly
Positive Control **
2
Concentration Range of DNA fragments
02 - .5 pmols*
.2 - 1.0 pmols*
0 pmols
3
Total Volume of Fragments (μl)
10
4
Gibson Assembly Master Mix (2x) (μl)
10
10
10
5
Deionized Water (μl)
10
10
0
6
Total Volume (μl) ***
20
20
20
Table1
*Optimized cloning efficiency is 50–100 ng of vectors with 2–3 fold of excess inserts. Use 5 times more of inserts if size is less than 200 bps. Total volume of unpurified PCR fragments in Gibson Assembly reaction should not exceed 20%.
** Control reagants are provided for 5 experiments with the Gibson Asembly Kit.
*** If greater numbers of fragments are assembled, additional Gibson Assembly Master Mix may be required.
Incubate samples in a thermocycler at 50°C for 15 minutes when 2 or 3 fragments are being assembled or 60 minutes when 4-6 fragments are being assembled.
Note: Extended incubation up to 60 minutes may help to improve assembly efficiency in some cases (for further details see FAQ section).
*Select the pencil icon and change the timer to 60 minutes if working with 4-6 fragments
00:15:00
Store samples on ice or at –20°C for subsequent transformation.
Purpose: To add the prefix and suffix fragments to the addgene insert to complete exon 1 assembly.
Materials:
NEB Hifi Assembly Kit
Addgene Insert with adapter overhang (previously PCRed)
IDT exon 1 flanks (previously restricted)
Water
A
B
C
1
Concentration
Volume
2
Prefix Fragment
3
Suffix Fragment
4
Addgene Insert
5
Total Volume of Fragments
6
Gibson Assembly Master Mix (2x) (μl)
7
Deionized Water (μl)
8
Total Volume (μl) ***
Table1
*Note: Experiment postponed bc previous digested exon flank was missing upon review of the digestion protocol it seems it was the restriction enzymes chosen were wrong. As StuI cuts exon flank in two places.
Gibson Assembly® Protocol (E5510)
Introduction
This is the protocol for Gibson Assembly using the Gibson Assembly® Cloning Kit (E5510). More information from NEB can be found here.
LB (Luria-Bertani) plates with appropriate antibiotic
Procedure
Set up the following reaction on ice:
Reaction volumes: Use this table to calculate reaction volumes and set up the reaction. Remember to input your total DNA fragment volume in cells B3 and C3 for assemblies with 2-3 fragments and 4-6 fragments, respectively.
- NEB recommends a total of 0.02–0.5 pmols of DNA fragments when 1 or 2 fragments are being assembled into a vector and 0.2–1.0 pmoles of DNA fragments when 4–6 fragments are being assembled. Efficiency of assembly decreases as the number or length of fragments increases. To calculate the number of pmols of each fragment for optimal assembly, based on fragment length and weight, we recommend using NEB's online tool, NEBioCalculator.
- The mass of each fragment can be measured using the NanoDrop instrument, absorbance at 260 nm, or estimated from agarose gel electrophoresis followed by ethidium bromide staining.
A
B
C
D
1
2-3 Fragments Assembly
4-6 Fragments Assembly
Positive Control **
2
Concentration Range of DNA fragments
02 - .5 pmols*
.2 - 1.0 pmols*
0 pmols
3
Total Volume of Fragments (μl)
10
4
Gibson Assembly Master Mix (2x) (μl)
10
10
10
5
Deionized Water (μl)
10
10
0
6
Total Volume (μl) ***
20
20
20
Table1
*Optimized cloning efficiency is 50–100 ng of vectors with 2–3 fold of excess inserts. Use 5 times more of inserts if size is less than 200 bps. Total volume of unpurified PCR fragments in Gibson Assembly reaction should not exceed 20%.
** Control reagants are provided for 5 experiments with the Gibson Asembly Kit.
*** If greater numbers of fragments are assembled, additional Gibson Assembly Master Mix may be required.
Incubate samples in a thermocycler at 50°C for 15 minutes when 2 or 3 fragments are being assembled or 60 minutes when 4-6 fragments are being assembled.
Note: Extended incubation up to 60 minutes may help to improve assembly efficiency in some cases (for further details see FAQ section).
*Select the pencil icon and change the timer to 60 minutes if working with 4-6 fragments
00:15:00
Store samples on ice or at –20°C for subsequent transformation.
Poured 1.5 mL of the culture into a microcentrifuge tube.
3.
Spun in microcentrifuge tube for 3 min at room temp. Placed in 4 degrees Celsius - store unused portion of the original culture at 4 degreees Celsuis
4.
Removed media w/ aspiration, leaving bacterial pellets as dry as possible
5.
Resuspended bacterial pellet in 200 mL of ice cole alkaline lysis solution 1 with vigorous vortexing (by hand)
6.
Added 250μl of freshly prepared alkaline lysis solution II to each bacterial suspension. Closed the tube tightly and mixed the contents by inverting the tube rapidly five times. Did not vortex - place the tube on ice.
Creating Exon 1 Fragment via PCR
Project: Lab Notebook
Authors: jason fuller
Date: 2017-10-23
Monday, 10/23/17
Purpose:
To couple IDT flanking sequences to AddGene HTT sequences.
Materials
See proceedure for PCR specific reagents
Template DNA:
PCRed out and Adapted AddGene Sequence
Exon1_flank_XmnI-Prefix
Exon1_flank-Cac8I-Adapter
Primers:
HTT-Assembly-Fwd
HTT-Assembly-REV
Procedure:
Follow protocol below
Flanking Region PCR Reaction
Calculations:
Previous gel of AddGene HTT sequences were compared to the NEB 2 log ladder.
●
All amplified parts appeared to coorespond to approximately 100 ng on gel. Gel lanes were loaded with 10ul of sample, thus 10 ul of each sample = ~100ng.
image.png
Flanking region DNA total = 1 pg in 50 ul
●
Therefore, 5 ul = 100 ng
Primer Dilution:
●
Starting quantity = 35 nM diluted into 100 ul
○
350 uM/L = stock primer concentration
●
1 ul of stock primer into 34 ul of H2O = 10 uM
○
1.25 ul of diluted stock = 0.5uM when diluted into a 25ul total reaction volume.
dNTP calculations
●
10 mM starting Conc.
○
dilute 1:2 to get 5mM
Taq Pol Calculations
●
5 U/uL starting Conc.
○
dilute 1:10 to get 0.5 U/uL
Anneling tempature calculations:
image.png
Elongation Calculations (1min / 1Kb)
●
Size of AddGene insert + AddGene Primer overlap =
Records of Quantities Used:
A
B
C
1
Componet
Volume (uL)
Final Conc
2
AddGene HTT DNA
9
~ 90 ng
3
cut IDT Flaking DNA
9
90 ng
4
10x PCR buffer
2.5
1 X
5
5mM dNTP Mix
1
0.2 mM (each)
6
10 uM forward primer
1.25
0.5 uM
7
10 uM reverse primer
1.25
0.5 uM
8
0.5 U/uL Taq Polymerase *
1
2.5 U / rxn
9
Water
0
n/a
Table1
*Add TaqPol after intial denatuartion at 95˚C for 10 min
PCR Protocol As Implemented:
1.
Intially denature dna at 95˚C for 10 min (with no polymerase)
2.
Add Polymerase
3.
Cycle 20x as follows
A
B
C
1
Tempature
Time
Cycle name
2
95˚C
45 s
Melt
3
65˚C
45 s
Anneal
4
72˚C
1 min / kb
Elongate
Table2
●
Cycle Name: JHF
3. End cycle as follows
A
B
C
1
72˚C
10 min
Final Elongation
2
4˚C
Indefinitly
Hold Chilled
PCR Finalization Parameters
●
Cycle Name: 95C-10M
4. Store DNA at -20˚C
A
B
C
1
Tempature
Time
Cycle name
2
95˚C
45 s
Melt
3
Tm - 5˚C (https://tmcalculator.neb.com/)
45 s
Anneal
4
72˚C
1 min / kb
Elongate
Post Hot start cycle values
●
Cycle Name: JHF
3. End cycle as follows
A
B
C
1
72˚C
10 min
Final Elongation
2
4˚C
Indefinitly
Hold Chilled
Final PCR steps
●
Cycle Name: 95C-10M
4. Store DNA at -20˚C
Expected Product (with varied CAG repeats):
HTT WildType Exon1 - Full
DNA Ladders
Project: HD Resolution iGEM Notebook
Authors: HD Resolution iGEM
Date: 2017-10-24
Tuesday, 10/24/17
NEB Quick Load Purple 2-Log Ladder
image.png
Thermoscientific Gene Ruler 1kb DNA Ladder
image.png
Invitrogen 10bp DNA Ladder
image.png
image.png
Amplification of Gibson Assembly Fragments
Project: HD Resolution iGEM Notebook
Authors: jason fuller
Date: 2017-10-24
Tuesday, 10/24/17
Purpose:
To amplify paired G-blocks (Blocks 1-3, 4-5, 6-7, 8-9, 10-11, 12-13, 14-15) from the Friday's lab.
Protocol:
The following protocol was followed:
Q5 Hi Fi 2X Master Mix Protocol
Additionally:
1.
All dry primer stocks were diluted to 500uM (0.5 nMoles/ µL).
2.
Diluted primer stocks to a 'working primer solution' with conenctration of 12.5 uM (1ul stock + 39 water)
3.
New PCR tubes were labled for each previous assembly.
4.
A 25 ul Reaction volume was chosen:
5.
Reagents were added as follows
A
B
1
Component
25 μL Reaction
2
Q5 High-Fidelity 2X Master Mix
12.5 μL
3
10 μM Forward Primer
1 μL
4
10 μM Reverse Primer
1 μL
5
Template DNA
5 ul + 5 ul
6
Nuclease-Free Water
5.5 ul *
Table1
* this volume was reduced by 2ul for the 1-3 reaction as 2 more primers were added.
Calculations
Primer dilution:
We need a final concentration of 0.5 uM in 25 uL. The minimum we can add with our pipets is 1 ul.
Thus we need a working solution containing 25x the concentration of 0.5 uM, or a 12.5 uM solution.
To get a 12.5uM solution from a 500uM stock, we need to dilute by a factor of 40. (500/12.5)
Thus we need 1ul of stock in 39 of water.
Primers Tms
2 Fwd & 3 Rev
image.png
2 Rev & 3 Fwd
image.png
4 Fwd & Rev
image.png
5 Fwd & Rev
image.png
6 Fwd & Rev
image.png
7 Fwd & Rev
image.png
8 Fwd & Rev
image.png
9 Fwd & Rev
image.png
All but 9 seem to anneal at 72, so we will use 72.
Visualization of PCR Exon 1 Fragments
Project: HD Resolution iGEM Notebook
Authors: jason fuller
Date: 2017-10-24
Tuesday, 10/24/17
Purpose: To run a gel electrophoresis to visualize the PCR amplicons of the full Exon 1 Assembly
1.
Split 10 lanes from precast 2% agarose gel
2.
Transfer 10ul from PCR tube to reaction tube
3.
Add 6x purple loading dye to each sample (2ul)
4.
Load 2% gel as follows:
A
B
C
D
E
F
G
H
I
J
K
1
Lane #
1
2
3
4
5
6
7
8
9
10
2
Sample
2 log ladder
1: MP Taq 24061
2: MP Taq 24061
3: Sat 24061
4: Invitrogen 24062
J1: MP Taq 24061
J2: MP Taq 24061
J3: Sat 24061
J4: Invitrogen 24062
2 log ladder
Table1
2 log ladder will resolve as follows:
image.png
4. Run gel at 100 volts for 30 min
5. Stain with ETBR with GENTLE shaking
6. Image using gel imager
RESULTS
Final days time calculaton: How Much Time do we need?
Project: HD Resolution Shared Project
Authors: jason fuller
Date: 2017-10-25
Wednesday, 10/25/17
Working Backwards:
Shipping the part: (7 hrs)
MiniPrep the Plasmid out of the cells = 1 hr
Dry the DNA (in 2 fractions) = 30 min
Reconstitute 1 of 2 fractions in water and take OD reading 10 min
Reconsititue remaning fration in ethanol to a concentration of 25ng/ul and transfer 10ul to shipping plate, then drying = 4-6 hrs
(I think we can do this in 1)
Add foil seal, pack, and get to a shipper 20 min
Unplanned Gotchas:
Guessing the pakage weight and getting and printing a shipping label with tracking number
People using the nanodrop when we go to take OD readings
Genewiz shipping number and sample tracking info needed to be submitted along with part before this point!
Cell Culture outgrowth: (19hrs) - 26
Pick colonies (that glow? that don't glow? how can we ensure we have the right plasmid?): 1 hr
Grow cultures over night: 16- 20 hrs
Unplanned Gotchas:
Not having sterlilized Chloramphenical containing media
Colonys too small
Colonies hard to differentatiate (no flourescense? - add 2 hrs?)
Transform and Grow Colonies (19 hrs) - 45
Prep and Transform colonies with Plasmid containing our Part: 1 hr
Grow colonies over night: 16- 20 hr
Ligate part into backbone and isolate
Order Oligos
Arrive noon tomorrow: 28 HRs 28 +45 = 6
Notes:
Separating supercoild plasmid dna and nicked plasmids from linear can be done using λ endonuclease (it can't restrict nicked plasmids) and RecJf https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2832929/
image.png
G-block 5-way Assembly
Project: HD Resolution iGEM Notebook
Authors: jason fuller
Date: 2017-10-25
Wednesday, 10/25/17
Purpose: Begin gibson assembly so that we can PCR once primers are in.
Materials:
IDT G-blocks (15)
NEB Hifi Builder Kit
Nuclease free water
A
B
C
D
1
Concentration Range of DNA fragments
02 - .5 pmols*
.2 - 1.0 pmols*
0 pmols
2
Total Volume of Fragments (μl)
10
3
Gibson Assembly Master Mix (2x) (μl)
10
10
10
4
Deionized Water (μl)
10
10
0
5
Total Volume (μl) ***
20
20
20
Table1
1.
Normalized concentrations of all G-blocks to 10 fmol per microliter.
2.
Take 5 microliters of each G-block sample and put into a new tube of each paired G-block sample.
3.
Then add 10 microliters of Gibson assembly master mix to each tube.
4.
Incubate for 15 minutes at 50 C in the thermocycler.
Gibson Assembly® Protocol (E5510)
Introduction
This is the protocol for Gibson Assembly using the Gibson Assembly® Cloning Kit (E5510). More information from NEB can be found here.
LB (Luria-Bertani) plates with appropriate antibiotic
Procedure
Set up the following reaction on ice:
Reaction volumes: Use this table to calculate reaction volumes and set up the reaction. Remember to input your total DNA fragment volume in cells B3 and C3 for assemblies with 2-3 fragments and 4-6 fragments, respectively.
- NEB recommends a total of 0.02–0.5 pmols of DNA fragments when 1 or 2 fragments are being assembled into a vector and 0.2–1.0 pmoles of DNA fragments when 4–6 fragments are being assembled. Efficiency of assembly decreases as the number or length of fragments increases. To calculate the number of pmols of each fragment for optimal assembly, based on fragment length and weight, we recommend using NEB's online tool, NEBioCalculator.
- The mass of each fragment can be measured using the NanoDrop instrument, absorbance at 260 nm, or estimated from agarose gel electrophoresis followed by ethidium bromide staining.
A
B
C
D
1
2-3 Fragments Assembly
4-6 Fragments Assembly
Positive Control **
2
Concentration Range of DNA fragments
02 - .5 pmols*
.2 - 1.0 pmols*
0 pmols
3
Total Volume of Fragments (μl)
10
4
Gibson Assembly Master Mix (2x) (μl)
10
10
10
5
Deionized Water (μl)
10
10
0
6
Total Volume (μl) ***
20
20
20
Table1
*Optimized cloning efficiency is 50–100 ng of vectors with 2–3 fold of excess inserts. Use 5 times more of inserts if size is less than 200 bps. Total volume of unpurified PCR fragments in Gibson Assembly reaction should not exceed 20%.
** Control reagants are provided for 5 experiments with the Gibson Asembly Kit.
*** If greater numbers of fragments are assembled, additional Gibson Assembly Master Mix may be required.
Incubate samples in a thermocycler at 50°C for 15 minutes when 2 or 3 fragments are being assembled or 60 minutes when 4-6 fragments are being assembled.
Note: Extended incubation up to 60 minutes may help to improve assembly efficiency in some cases (for further details see FAQ section).
*Select the pencil icon and change the timer to 60 minutes if working with 4-6 fragments
00:15:00
Store samples on ice or at –20°C for subsequent transformation.
Purpose: Reassemble and cut existing Exon 1 parts such that there is a definite Eco R1 restriction site so that they can be added into a pSB1C3 backbone
1.
Placed previously isolated DNA plasmid on ie (61 and 62)
2.
Measured the concentration of the DNA Plasmids with the nanodrop (2μl)
a.
DNA plasmid 40261 : 356.2 ng/μl
b.
DNA plasmid 40262 : 300.5 ng/μl
3.
Diluted DNA plasmid 1 : 3 to approximate 100 ng/μl and measured with nanodrop
a.
DNA plasmid 40261 : 117.9 ng/μl
b.
DNA plasmid 40262 : 106.1 ng/μl
4.
Labeled fresh 0.5 mL tubes 61 and 62
5.
For each tube, transferred 4 ul of 10x NEBuffer, 1 ul of Eco R1, 1 ul of Xmn 1, and 25.6 ul of dH2O*
6.
Transferred 10μl DNA Plasmid 40261 to 0.5 mL tube 61 and 10μl of DNA plasmid 40262 to 0.5 mL tube 62
7.
Incubated 0.5 mL tubes at 37 degrees Celsius for fifteen minutes
8.
Added 10μl purple 6x loading dye to each of the 0.5 mL tube and mixed
9.
Gel was loaded as follows:
A
B
C
D
E
F
G
1
Cane
1
2
3
4
5
6
2
10μl of 2 log ladder
10μl of digested ladder
10μl of digested 40262
15μl of digested 40262
15μl of digested 40261
15μl of digested 40261
10μl of 2 log ladder
Table1
*NOTE: NEBuffer and H2O was accidentally pippeted into the DNA Plasmids (tubes 61 and 62)
- So 18 ul of each 61 and 62 were pipetted into corresponding 61 and 62 tubes (the other ones)
