Difference between revisions of "Team:Georgia State/notebookdata.html"
| Line 836: | Line 836: | ||
<div id="date-10-14"> | <div id="date-10-14"> | ||
<h1>Saturday, October 14th.</h1> | <h1>Saturday, October 14th.</h1> | ||
| − | <li>Eudoxie Bataba</li> | + | <li>Eudoxie Bataba</li> |
<ul><b>Objective:</b>PCR diagnostic gel, Miniprep and Restriction DIgest</ul> | <ul><b>Objective:</b>PCR diagnostic gel, Miniprep and Restriction DIgest</ul> | ||
<ul><b>Materials:</b>QIAGEN miniprep kit, 50% glycerol</ul> | <ul><b>Materials:</b>QIAGEN miniprep kit, 50% glycerol</ul> | ||
Revision as of 14:11, 1 November 2017
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Team:Tec-Monterrey/ITESM14 notebook data.html - 2014.igem.org
Tori Radcliff
Tori Radcliff
Tori Radcliff
Monday, May 1st.
Sunday, June 1st.
Monday, June 2nd.
Tuesday, June 3rd.
Wednesday, June 5th.
Thursday, June 5th.
Friday, June 6th.
Saturday, June 7th.
Sunday, June 8th.
Monday, June 9th.
Tuesday, June 10th.
Wednesday, June 11th.
Thursday, June 12th.
Friday, June 13th.
Saturday, June 14th.
Sunday, June 15th.
Monday, June 16th.
Tuesday, June 17th.
Wednesday, June 18th.
Thursday, June 19th.
Friday, June 20th.
Saturday, June 21st.
Sunday, June 22nd.
Monday, June 23rd.
Tuesday, June 24th.
Wednesday, June 25nd.
Thursday, June 26th.
Friday, June 27th.
Saturday, June 28nd.
Sunday, June 29nd.
Monday, June 30nd.
Tuesday, July 1st.
Wednesday, July 2nd.
Thursday, July 3rd.
Friday, July 4th.
Wednesday, July 5th.
Thursday, July 6th.
Friday, July 7th.
- Objective:To determine if Alkaline Phosphotase will bind to nitrocellulose paper and produce a signal when mixed with BCIP.
- Materials:Cut small strips of nitrocellulose paper (Nitran), Promega Alkaline Phosphatase (Conc: 1ng/ul), Alkaline 10X buffer diluted, BCIP
- Equipment:UV-Crosslinker
- Procedure:See attached procedure.
- Results: The Ap spread outward but stayed confined meaning the crosslinking worked. Adding the BCIP solution to the tube with the paper produced a noticeable discoloration. Adding the enzyme to the BCIP only produced a small discoloration. The binding of the alkaline phosphatase and BCIP to the paper was successful. The reaction worked only in the intended areas meaning that if the AP is bound to the factor c it may produce a signal only if it is cut, the AP nor BCIP disperses in the solution after being bound to the paper.
Saturday, July 8th.
Sunday, July 9th.
Monday, July 10th.
Tuesday, July 11th.
Wednesday, July 12th.
Thursday, July 13th.
Friday, July 14th.
Saturday, July 15th.
Sunday, July 16th.
Monday, July 17th.
Tuesday, July 18th.
Wednesday, July 19th.
Thursday, July 20th.
Friday, July 21st.
Saturday, July 22nd.
Sunday, July 23rd.
Monday, July 24th.
Tuesday, July 25th.
Wednesday, July 26th.
Thursday, July 27th.
Friday, July 28th.
Saturday, July 29th.
Sunday, July 30th.
Monday, July 31st.
Tuesday,August 1st.
- Objective:Before the fusion step, the hCG needs to be placed into the PSB1C3 plasmid, so that the hCG can be submitted. In order to add the hCG to add to the plasmid restriction sites need to be added this was done by adding the prefix and suffix through PCR.
- Materials:Qiagen Master mix (2x), Prefix Primer, Suffix Primer, hCG DNA (diluted to 10ng/ul), and Nuclease free H20
- Equipment:Thermocycler
- Procedure:See attached procedure.
- Results:
 |  |
| Simulated gel using SnapGene | 1% Agarose gel, experimental results |
Wednesday, August 2nd.
Thursday, August 3rd.
- Objective:To double digest both the PSB1C3 backbone and our hCG PCR product
- Materials:DNA of ECFP-PCB1C3 (102 ng/ul) and hCG PCR product, Restriction enzymes: NotI, EcoRI, XbaI, PstI,SpeI, Green Fastdigest Buffer (10x), and Nuclease free H20
- Equipment:Thermocycler
- Procedure:See attached procedure.
- Results:
- 1. 1Kb New England Biolabs Ladder
- 2. hCG uncut
- 3. hCG cut with EcoR1 and Pst1
- 4. hCG cut with Xba1 and Spe1
- 5. ECFP cut with EcoR1 and Pst1
- 6. ECFP cut with Xba1 and Spe1
 |  |
| Simulated gel using SnapGene | The Wells Contain the following:
|
Friday August 4th.
- Objective: Clean used GST resin so that it may be used in future protein purifications.
- Materials: GST Resin, 70% Ethanol, GST Elution Buffer
- Equipment: Centrifuge,Vortexer, Micropipette,Eppendorf tubes/Falcon tubes, Spectrophotometer, and Cuvettes
- Procedure:See attached procedure for buffer recipes and instructions.
- Results:
| Sample | Absorbance @ 280 nm |
|---|---|
| Clean GST Elution Buffer (Blank) | 0 |
| Elution 1 | 0.667 |
| Elution 2 | 0.036 |
Saturday, August 5th.
- Objective:To repeat the double digest of PSCB1C3 and hCG PCR
- Materials:DNA of Lt10-PCB1C3 (80 ng/ul) and hCG PCR product, Restriction enzymes: NotI, EcoRI, XbaI, PstI,SpeI, Green Fastdigest Buffer (10x), and Nuclease free H20
- Equipment:Thermocycler
- Procedure:See attached procedure.
- Results:
| Since the diagnostic gel was a success the PSB1C backbone was extracted from the Lt 10 plasmid using an E-gel. |
Sunday, August 6th.
Monday, August 7th.
- Objective:Culture of transformed E.coli with mamba in PGEX
- I picked 10 colonies from plates and did overnight cultures in LB
Tuesday, August 8th.
- Objective:I will get the mamba in PGEX plasmid from transformed E.coli
- Materials:NEB miniprep kit, 50% glycerol
- Equipment:Centrifuge and Nanophotometer
- Procedure:See attached procedure.
- Results:
| Sample | Concentration (ng/uL) |
|---|---|
| 1 | 118 |
| 2 | 123 |
| 3 | 85 |
| 4 | 133 |
| 5 | 90 |
| 6 | 75 |
| 7 | 72 |
| 8 | 100 |
| 9 | 65 |
| 10 | 140 |
Wednesday, August 9th.
- Ligation of hCG to PSB1C3
Thursday, August 10th.
- Transformation of hCG-PSB1C3 ligation and overnight culture
Friday, August 11th.
- Colony PCR of hCG-PSB1C3 colonies
Saturday, August 12th.
Sunday, August 13th.
- Overnight culture of hCG-PSB1C3 colonies
Monday, August 14th.
- Miniprep of hCG-PSB1C3 cultures
- Diagnostic restriction digest
- PCR of factor-C gBlocks
- Objective: Factor C was divided into 4 G-blocks and ordered from IDT. A PCR was performed to add overlaps to each block so that subsequent overlap extension PCRs could be performed.
- Procedure: See attached protocol.
- Results:
 |
| A simulated gel was created and then the actual sampled of each were run on an e-gel. 5ul of product with 1ul of 6X dye and 14 ul of DI water was added to each well. The wells include the following from left to right: Gene Ruler 1kb DNA ladder, PCR of g-block 1, PCR of g-block 2, PCR of g-block 3, PCR of g-block 4. |
Tuesday, August 15th.
- Overlap Extension PCR
- Objective:To combine the four PCR-ed g-blocks of factor-C.
- Procedure: See attached protocol for details
- Results:
 |  |
| Simulated gel | Wells contain the following: Gene Ruler 1kb plus MW ladder, Reaction of blocks 1 and 2, Negative control (blocks 1 and 2), Reaction of blocks 3 and 4, Negative control (blocks 3 and 4), Reaction of 1 and 2 with 3 and 4 products, negative control (reactions 1 and 2 with 3 and 3 and 4), and Gene Ruler 1kb plus MW ladder. |
Wednesday, August 16th.
Thursday, August 17th.
- Objective:Prepare hCG for ligation into pGEX through PCR
- Materials:hCG DNA, primers forward and reverse and master mix
- Equipment:Thermocycler
- Procedure:Two 50 ul PCR reactions were performed. 25ul of qiagen mastermix, 2ul of block 5 DNA, 2.5ul of forward primer, 2.5 ul of reverse primer and 18 ul of nuclease-free water was added to tube 1 and tube 2.
- Overnight cultures
- Colony PCR of Cara's transformants
Friday, August 18th.
Saturday, August 19th.
Sunday, August 20th.
Monday, August 21st.
- Miniprep
- Ligations
Tuesday, August 22nd.
Wednesday, August 23rd.
Thursday, August 24th.
Friday, August 25th.
- Objective:To repeat the double digest of PSCB1C3 and hCG PCR
- Materials:DNA of Lt10-PCB1C3 (80 ng/ul) and hCG PCR product, Restriction enzymes: NotI, EcoRI, XbaI, PstI,SpeI, Green Fastdigest Buffer (10x), and Nuclease free H20
- Equipment:Thermocycler
- Procedure:See attached procedure.
Saturday, August 26th.
Sunday, August 27th.
Monday, August 28th.
Tuesday, August 29th.
- Ligation of hCG and PSB1C3
- Objective:To ligate hCG and PSB1C3
- Materials: digested vector cleaned by Cara Jones, T4 ligase and buffer, digested hCG
- Procedure: Using a ligation calculator the concentration needed was 31.87 ng/ul in a 20ul total ligation. 1ul of PGEX at 105 ng/ul and 6 ul of hCG, 2 ul of T4 buffer, 1 ul of T4 ligase and 10 ul of nuclease-free water. A negative control was used which included digested mamba in pgex with no ligation buffer. The ligation reaction sat at room temperature for 10 minutes.
- Transformation
- Objective:To transform the ligation of hCG and PSB1C3
- Materials:C2987I NEB competent cells, ligation reaction
- Equipment:shaker and incubator
- Procedure:See attached procedure.
- Results:There was no growth on the ligation plate nor negative control but there was growth on the positive control. Since there was no growth on the ligation plates that would mean the cells have no resistance to ampicillin and the ligation was inefficient due to the fact the hCG was not purified to eliminate contaminants ike EDTA and salts.
Wednesday, August 30th.
Thursday, August 31st.
Friday, September 1st.
Saturday, September 2nd.
Sunday, September 3rd
Monday, September 4th
Tuesday, September 5th
Wednesday, September 6
Thursday, September 7
Friday, September 8th.
- hCG for pGEX pcr
- Results:
 |
| test |
Saturday, September 9
Sunday, September 10
Monday, September 11
Tuesday, September 12
Wednesday, September 13
Thursday, September 14th.
Friday, September 15
Saturday, September 16th.
Sunday, September 17
Monday, September 18
Tuesday, September 19
Wednesday, September 20
Thursday, September 21
Friday, September 22
Saturday, September 23
Sunday, September 24
Monday, September 25
Tuesday, September 26
Wednesday, September 27
Thursday, September 28
Friday, September 29
Saturday, September 30
Sunday, October 1st.
Monday, October 2nd.
Tuesday, October 3rd.
Wednesday, October 4th.
Thursday, October 5th.
Friday, October 6th.
Saturday, October 7th.
Sunday, October 8th.
Monday, October 9th.
Tuesday, October 10th.
Wednesday, October 11th.
Thursday, October 12th.
- I transformed HCG in PS1C3 into E.coli today
Friday, October 13th.
- I performed PCR and did overnight cultures of 10 colonies
Saturday, October 14th.
- Objective:PCR diagnostic gel, Miniprep and Restriction DIgest
- Materials:QIAGEN miniprep kit, 50% glycerol
- Equipment:Centrifuge, Nanophotometer, Thermocycler
- Procedure:See attached procedure.
- Result:See attached PCR results.
Sunday, October 15th.
- Restriction digests
- Ligation
Monday,October 16th.
- Transformations
Tuesday, October 17th.
- Ligation
Wednesday,October 18th.
- Digested miniprepped DNA from glycerol stocks
- Gel extraction of the backbones
Thursday, October 19th
- Double digests
- Ligations
- Transformations
Friday, October 20th
- Colony pcr
 |
 |
Saturday, October 21st
- Miniprep colonies
Sunday, October 22nd.
Monday, October 23rd.
- Colony PCR
- Diagnostic restriction digest of colonies
- Gel extraction and clean-up
Tuesday, October 24th.
- Digested three different plasmids for ligation with HCG. All were PSB1C3 with different inserts.
- Gel extraction and clean-up
Wednesday, October 25th.
- Minipreps of colonies from the plates from 10/19
- Restriction digests
Thursday, October 26th.
- Diagnostic gel
- Minipreps
Friday, October 27th.
DNA Submission Deadline!!Saturday, October 28th.
Sunday, October 29th.
Monday, October 30th.
- Freak out about our wiki.
Tuesday, October 31st.
- Freak out about our wiki.
Wednesday, November 1st.
WIKI FREEZE!!!!!!- Freak out about our wiki.
