Team:Aachen/Protocols

iGEM Team Aachen 2017

Protocols

General:

- This protocol was designed to isolate mRNA from a yeast culture and perform a RT-qPCR for proofing gene expression of a specific gene.
- When working with mRNA only use nuclease-free chemicals and materials. Wear gloves and change them frequently. Do not talk to prevent the mRNA from RNases
- Protocol for mRNA isolation was taken from “RNaesy Mini Handbook” (Qiagen). Respective chemicals and buffers are included in this kit.

Specific for our use:

- Following strains were tested:
o Wildtype
o AVP1-mutant with not induced promotor (grown in YPD with glucose)
o AVP1-mutant with induced promotor (grown in YPD with galactose)
- mRNA-isolation and all steps except of RT-qPCR were performed in duplicates per strain
- RT-qPCR itself was performed in triplicates per sample
- For reference we used a housekeeping gen. We chose the yeast gene ACT1.
- Primer pairs used in the RT-qPCR are designed to amplify a gene fragment which is 100 bases in length within the AVP1 gene und the housekeeping gene ACT1 each

Preparation of cells:

- Inoculate 10 ml YPD containing 100% galactose and 10 ml normal YPD for reference with a cryo-culture of yeast cells and let it growth at 28 C°, 150 rpm for 12-16 hours (preculture)
- Inoculate 50 ml YPD containing 100% galactose or normal YPD with 2 ml of preculture and let it grow until OD600 0,4

Cell lysis and mRNA-isolation

- Media complementation right before cell lysis
o Add 0,1% β-mercaptoethanol and zymolyase to prepared Y1-buffer-stock-solution
 Recommendations: For cell lysis of 50 ml culture (OD600 0,4) you will need 2ml Y1-stock-solution, 2 µl β-mercaptoethanol and 5 mg zymolyase (20800 units)
- Centrifuge cells in 50 ml flask at 1000xg, 4C° for 5 min
- Discard supernatant
- Resuspend cells in 100 µl nuclease-free-water and transfer to a 2 ml Eppendorf-reaction-tube
- Centrifuge at 1000xg for 1 min
- Discard supernatant
- Resuspend cells in 2 ml Y-buffer and incubate at 35 C°, 300 rpm for 20 min (conditions depending on the enzyme which is used)
- Centrifuge cells at 300 rpm for 5 min to sediment originated spheroplasts
- Discard supernatant
- Add 350µl buffer RLT and vortex vigorously to destroy spheroplasts
- Add 350µl ethanol (70% (v/v)) and mix while pipetting
- Transfer solution to an RNeasy-column siting in 2ml tube
- Close lid and centrifuge at 8000xg for 15 s
- Discard supernatant
- Add 700µl buffer RW1 to the column, centrifuge at 8000xg for 15s
- Discard supernatant
- Add 500µl buffer RPE and centrifuge at 8000xg for 15s
- Discard supernatant
- Add 500µl RPE and centrifuge at 8000xg for 2min
- Place column in a new tube and centrifuge at full speed for 1min
- Discard supernatant
- Place column in a new tube and add 40µl nuclease-free-water
- Centrifuge at 10000 rpm for 1min
- Discard column

Preparation of RT-qPCR

- Measure mRNA concentration in eluate
- Extract 3 µg of mRNA (respective µl) and place in nuclease-free Eppendorf reaction tube for DNA digestion
- Add 3µl DNaseI, 3 µl Dnase buffer and fill up to 30µl with nuclease-free water
- Incubate mixture at 37 C° for 30min
- Add 3 µl EDTA (25 mM)
- Incubate at 75 C° for 10min
- Extract 11 µl (=1µg) mRNA-mixture and place in a nuclease-free Eppendorf reaction tube, add 14 µl nuclease-free water and 2 µl primer (0,2µM; dNTP-primer for binding at polyadenylation-sequence of mRNA; here olio dTprimer PVX)
- Incubate at 80 C° for 10min
- Cool on ice shortly
- Transfer mixture to a pcr-tube
- Add 1 µl M-MLV (100 Units, reverse transcriptase), 4µl 5X M-MLV-buffer, 0,5mM dNTPs and fill up to 50µl with nuclease-free water
- Perform a pcr with following conditions:
o 1. 40 C° for 30min
o 2. 45 C° for 20min
o 3. 50 C° for 20min
o 4. 55 C° for 20min
o 5. 75 C° for 15min
o Conditions depend on length of mRNA
o No repetitions
- Add 1µl RNaseI and incubate at 37 C° for 1hour to digest RNA
- Prepare mixture for RT-qPCR
o Extract 5µl of mRNA mixture and transfer to Eppendorf reaction tube
o Add 300nM of each primer pair (primers for housekeeping gen and primers for gen to be verified)
o Add 10µl SYBr-Green
o Fill up to 20µl with ddH20
o Transfer your samples to 96 well-plate for RT-qPCR
- Perform a pcr with following conditions
o 1. 95 C° for 3min
o 2. 95 C° for 15s
o 3. 55 C° for 30s repeat 40 times
o 4. 72 C° for 30s
o Conditions depend on the properties of the used primers and on the length of the amplified sequence

- Perform a melting curve of the originated cDNA product to check the right amplification (conditions depend on the machine you are using)

Media:

- Buffer Y1-stock solution:
o 1M Sorbitol
o 0,1M EDTA
o Adjust the pH value to pH = 7,4 with HCL and NaOH and autoclave
 Example: To produce 50ml Y1-stock-solution:
9,109g Sorbit ( n = 1mol/L * 0,05 L = 0,05mol ; m = n * M = 0,05mol * 182,18g/mol = 9,109g)
1,8612g EDTA ( n = 0,1mol/L * 0,05 L = 0,005mol ; m = n * M = 0,005mol * 372,24g/mol = 1,8612g)
- Buffer Y1-working-solution
o Y1-stock-solution
o 0,1% β-mercaptoethanol and zymolyase (calculate 5mg zymolyase per 50 ml cell culture with OD600 0,4)

References:

- Script “Real Time PCR, Applications Guide” (Biorad)
- RNeasy Mini Handbook (Qiagen)
- Dissertation of Christina Dickmeis (RTWH Aachen)

To proof the transcription of our integrated AVP1-DNA, we performed a SDS-PAGE gel electrophoresis.

Protocol for preparation of cells:

- Prepare an overnight-culture of your cells in 10 ml YPD-media and incubate at 28 C° and 250 rpm

o We prepared four overnight-cultures:
 Wildtype (S. cerevisiae BY4742) in YPD-media
 AVP1-mutant in YPD-media
 AVP1-mutant in YPD-media
 AVP1-mutant in YPD-media with galactose instead of glucose

- After 12 hours of incubation inoculate 50 ml of YPD-media with the entire overnight-culture and let it grow at 28 C°, 250 rpm until OD600 0,4 is reached
o We inoculated as followed:
 Overnight-culture (1) we inoculated in 50 ml YPD
 Overnight-cuture (2) we inoculated in 50 ml YPD
 Overnight-culture (3) we inoculated in 50 ml YPD with galactose instead of glucose
 Overnight-culture (4) we inoculated in 50 ml YPD with galactose instead of glucose
- Calculate total OD-values: Multiple the measured with the culture volume (is needed later)
- Centrifuge cells in 50 ml flasks at 1000 xg, 4 C° for 5 min, discard supernatant and resuspend pellet in 50 mml cold water
- Centrifuge cells again (1000 xg, 5 min, 5 min) and discard supernatant
- Freeze cells at -80 C° or continue with “Protocol cell disruption and gel loading”

Protocol cell disruption and gel loading

- Heat “cracking buffer” at 60 C°
- Resuspend (frozen) cell pellet in cracking buffer (100 µl of cracking buffer per 7,5 total OD600)
- Place mixture in 1,5 ml screw cap centrifuge tubes, filled with 80 µl glass beads (0,5 mm diameter) per 7,5 total OD 600
- Heat sample at 70 C° for 10 min
- Vortex for 1 min
- Centrifuge cells at 14000 rpm, 4 C° for 5 min
- Place supernatant (1) on ice and continue protocol with pellet
- Heat pellet at 100 C° for 3-5 min (eventually add new cracking buffer if necessary)
- Vortex for min
- Centrifuge cells at 14000 rpm, 4 C° for 5 min
- Place supernatant on ice (2)
- Boil both supernatants (1 and 2) shortly (2 min) in water bath and immediately load SDS-gel
o We received eight samples and loaded on gel

Notes:

- Important: Add 100 x PMSF every 7 minutes during the procedure to inhibit proteases (Use 1 µl PMSF per 100 µl Cracking buffer
- All centrifugation steps were conducted in a benchtop centrifuge
- All heating steps were conducted in a heat block

Supplements of cracking buffer:
- Cracking buffer stock solution:

Chemicals - Final concentration - To prepare 100 ml
Urea - 8 M - 48g
SDS - 5 % m/v - 5g
Tris HCl (pH 6,8) - 40 mM - 4 ml of 1M stock solution
EDTA - 0,1 mM - 20 µl of 0,5 M stock solution
Bromphenol Blau - 0,4 mg/ml - 40 mg
ddH2O - To final volume of 100 ml

- Cracking buffer (used for protocol):
Cracking buffer stock solution - 1 ml
Beta-mercaptoethanol - 10µl
PMSF - 50µl
ddH2O - 70 µl

- 0,0348 g PMSF (solid) was solved in 2 ml isopropanol

Reference for this Protocol: “Yeast Protocol Handbook”

This protocol is designed for purification of up to 20 µg of high-copy plasmid DNA from 1-5ml overnight cultures of E.coli in LB medium. Here a purification kit from “QIAGEN” is used.

Note: All protocol steps should be carried out at room temperature (15-25*C).

Procedure:

1. Resusped pelleted bacterial cells in 250µl Buffer P1 and transfer to a microcentrifuge tube.

- Ensure that RNase A has been added to Buffer P1. No cell clumps should be visible after resuspension of the pellet.
- If LyseBlue reagent has been added to Buffer P1, vigorously shake the buffer bottle to ensure LyseBlue particles are completely dissolved. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.

2. Add 250 µl Buffer P2 and mix thorougly by inverting the tube 4-6 times.

- Mix gently by inverting the tube. Do not vortex, as this will result in shearing of genomic DNA. If necessary, continue inverting the tube until the solution becomes viscous an slightly clear. Do not allow the lysis reaction to proceed for more than 5 min.
- If LyseBlue has been added to Buffer P1 the cell suspension will turn blue after addition of Buffer P2. Mixing should result in a homogeneously colored suspension.
- If the suspension contains localized colorless regions of if brownish cell clumps are still visible, continue mixing the solution until a homogeneously colored suspension is achieved.

3. Add 350 µl Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.

- To avoid localized precipitation, mix the solution thoroughly, immediately after addition of Buffer N3. Large culture volumes (e.g. > 5ml) may require inverting up to 10 times. The solution should become cloudy.
- If LyseBLue reagent has been used, the suspension should be mixed until all trace of blue has gone and the suspension is colorless. A homogeneous colorless suspension incated that the SDS has been effectively precipitated.

4. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.

- A compact white pellet will form

5. Apply 800 µl pf the supernatant from step 4 to the QIAprep 2.0 spin column by pipetting.

6.Centrifuge for 30-60 s. Discard the flow-through.

7. Recommended: Wash the QIAprep 2.0 spin column by adding 0,5 ml Buffer PB and centrifuging for 30-60 s. Discard the flow-through.

- This step is necessary to remove trace nuclease activity when using endA+ strains such as the JM series, HB101 and its derivatives, or any wild-type strain, which have high levels of nuclease activity or high carbohydrate content. Host strains such as XL-1 Blue and DH5a do not require this additional wash step.

8. Wash QIAprep 2.0 spin column by adding 0,75 ml Buffer PE and centrifuging for 30-60 sec.

9. Discard the flow-through, and centrifuge at full speed for additional 1 min to remove residual wash buffer.

- important: Residual wash buffer will not be completely removed unless the flow-through is discarded before this additional centrifugation. Residual ethanol from Buffer PE max inhibit subsequent enzymatic reactions.

10. Place the QIAprep 2.0 column in a clean 1,5 ml microcentrifuge tube to elute DNA, add 50 µl Buffer EB (10mM Tris-Cl, pH 8.5) or water to the center of each QIAprep 2.0 spin column, let stand for 1 min, and centrifuge for 1 min.

1. Adjust DNA binding conditions

- For very small sample volumes <30 µl adjust the volume of the reaction mixture to 50-100 µl with water. It is not necessary to remove mineral oil.
- Mix 1 volume of sample with 2 volumes of Buffer NTI (e.g., mix 100 µl PCR reaction and 200 µl Buffer NTI).
- Note: For removal of small fragments like primer dimers dilutions of Buffer NTI can be used instead of 100% Buffer NTI. Please refer to section 2.3.

2. Bind DNA

- Place a NucleoSpin Gel and PCR Clean-up Column into a Collection Tube (2 ml) and load up to 700 µl sample.
- Centrifuge for 30 s at 11,000 x g. Discard flow-through and place the column back into the collection tube.
- Load remaining sample if necessary and repeat the centrifugation step.

3. Wash silica membrane

- Add 700 µl Buffer NT3 to the NucleoSpin Gel and PCR Clean-up Column. Centrifuge for 30 s at 11,000 x g. Discard flow-through and place the column back into the collection tube.
- Recommended: Repeat previous washing step to minimize chaotropic salt carry-over and improve A(260)/A(230) values (see section 2.7 for detailed information).

4. Dry silica membrane

- Centrifuge for 1 min at 11,000 x g to remove Buffer NT3 completely. Make sure the spin column does not come in contact with the flow-through while removing it from the centrifuge and the collection tube.
- Note: Residual ethanol from Buffer NT3 might inhibit enzymatic reactions. Total removal of ethanol can be achieved by incubating the columns for 2-5 min at 70°C prior to elution.

5. Elute DNA

- Place the NucleoSpin Gel and PCR Clean-up Column into a new 1,5 mL microentrifuge tube (not provided). Add 15-30 µl Buffer NE and incubate at room temperature (18-25°C) for 1 min at 11,000 x g.
- Note: DNA recovery of larger fragments (>1000 bp) can be increased by multiple elution steps with fresh buffer, heating to 70°C and incubation for 5 min. See section 3,6 for detailed information.

PCR-cleanup kit from “NucleoSpin” is used.

1. Excise DNA fragment/solubilize gel slice

Note: Minimize UV exposure time to avoid damaging the DNA.
Take a clean scalpel to excise the DNA fragment from an agarose gel. Remove all excess agarose.
Determine the weight of the gel slice and transfer it to a clean tube
For each 100 mg of agarose gel < 2% add 200 µL Buffer NTI.
For gels containing <2% agarose, double the volume of Buffer NTI.
Incubate sample for 5-10 min at 50°C. Vortex the sample briefly every 2-3 min until the gel slice is completely dissolved!

2. Bind DNA

Place a NucleoSpin Gel and PCR Clean-up Column into a Collection Tube (2 ml) and load up to 700 µl sample.
Centrifuge for 30 s at 11,000 x g. Discard flow-through and place the column back into the collection tube.
Load remaining sample if necessary and repeat the centrifugation step.

3. Wash silica membrane Add 700 µl Buffer NT3 to the NucleoSpin Gel and PCR Clean-up Column. Centrifuge for 30 s at 11,000 x g. Discard flow-through and place the column back into the collection tube.
Recommended: Repeat previous washing step to minimize chaotropic salt carry-over and improve A(260)/A(230) values (see section 2.7 for detailed information).

4. Dry silica membrane

Centrifuge for 1 min at 11,000 x g to remove Buffer NT3 completely. Make sure the spin column does not come in contact with the flow-through while removing it from the centrifuge and the collection tube.
Note: Residual ethanol from Buffer NT3 might inhibit enzymatic reactions. Total removal of ethanol can be achieved by incubating the columns for 2-5 min at 70°C prior to elution.

5. Elute DNA

Place the NucleoSpin Gel and PCR Clean-up Column into a new 1,5 mL microentrifuge tube (not provided). Add 15-30 µl Buffer NE and incubate at room temperature (18-25°C) for 1 min at 11,000 x g.
Note: DNA recovery of larger fragments (>1000 bp) can be increased by multiple elution steps with fresh buffer, heating to 70°C and incubation for 5 min. See section 3,6 for detailed information.

1. Setup on ice in a 0.2 ml tube the mastermix for PCR according to the table below. Thaw all non-enzyme components at RT, mix by short vortex and collect by short centrifugation. It is recommended to add PhuS DNA Polymerase last in order to prevent any primer degradation caused by the 3`→5`exonuclease activity. Polymerase may be diluted in 1 x Buffer just prior to use in order to reduce pipetting errors.

Mastermix: (50 µL reaction)

Plasmid template (sequencing or column grade plasmid preparation) - X µL (10-15 ng, max. 50 ng)
Deionized H20 (PCR quality) 35.5 µL - X µL of plasmid template
10 x PfuS buffer B2 - 5 µL (final 1x)
X_fwd primer [1] (5 µM) - 4 µL (final 400 nM)
x_rev primer (5 µM) - 4 µL (final 400 nM)
dNTP-mix (10mM each) - 05 1 µL (final 0.2 mM each nucleotide)
PfuS (2 U/µl [2] E2 - 0,5 µL (final 0.02 U/µL)

PCR programm: (25 cycles, tube control with 50 µL, 105 °C heated lid)

ID - Initial denaturation - 30 sec - 98°C
D - denaturation - 5-10 sec [3] 98°C - 24 cycles (D,A,E)
A - annealing - 15-30 sec - X °C (45-72°C) [4]
E - extension - 15-30 sec/Kb [5] - 72 °C
FE - final elongation [6] - 5-10 min - 72 °C
S - Storage - forever - 8°C

 optional 2.Load all samples on a 0,8% TEA agarose-gel (100V; 35 min). Check for optimal annealing temperature and if enough PCR product is present gel-extract the bands of column purifiy is with the PCR purification-kit. (Macherey-Nagel NucleoSpin Extract II; 50 µL Ne-buffer).

Notes

 [1] Primers: Oligonucleotides primers are generally 20-40 nucleotides in length and ideally have a GC content of 40-60%. The final concentration of each in a PCR reaction may be 0.2-1 µM. while 0.4 µM is recommended.

 [2] PfuS DNA polymerase concentration: recommended use of 1 U/50 µL reaction. However, the optimal concentration of PfuS DNA polymerase may vary from 0.5-2 U/50 µL reaction depending on amplicon length and difficulty. Do not exceed 2 U/50 µL reaction, especially for amplicons longer than 5 kb (https://www.neb.com).

 [3] Keep the denaturation as short as possible. Usually 5-10 seconds at 98°C is enough for most templates. Note: the denaturation time and temperature may vary depending on the ramp rate and temperature control mode of the cycler.

 [4] The PfuS DNA polymerase has the ability to stabilize primer-template hybridization. As a basic rule, for primers > 20 nt, anneal for 10-30 sec at a Tm + 3 °C of the lower primer. The Tm´s should be calculated with the nearest neighbor method (calculate tm for matching sequence with http://eu.idtdna/analyzer/applications/oligoanalyer/default.aspx) as results from primer Tm calculations can vary significantly depending on the method used. For primers <20 nt, use a annealing temperature equal to the Tm of the lower Tm primer.

 [5] Extension time depends on amplicon length and complexity. For low complexity DNA (e.g. plasmid, lambda or BAC DNA) use extension time of 15 sec per 1 kb. For high complexity genomic DNA 30 sec per 1 kb is recommended.

 [6] Final extension is an optimal step, usually does not influence the outcome and can by default be skipped.

reaction setup  We recommend assembling all reaction components on ice and quickly transferring the reactions to a thermocycler preheated to the denaturation temperature (94°C)

Component 25 µL reaction 50 µL reaction Final Concentration

10 µM Forward Primer 0,5 µL 1 µL 0,2 µM
10 µM Reverse Primer 0,5 µL 1 µL 0,2 µM
Template DNA variable variable <1,000 ng
One Taq Quick-Load 2X Master Mix with Standard Buffer 12,5 µL 25 µL 1X
Nuclease-free water to 25 µL to 50 µL

 Notes: Gently mix the reaction. Collect all liquid to the bottom of the tube by a quick spin if necessary.
Overlay the sample with mineral oil if using a PCR machine without a heated lid.
 Tranfer PCR tubes to a PCR machine and begin thermocycling:

Thermocycling conditions for a routine PCR:

STEP TEMP TIME

Initial Denaturation 94°C 30 seconds
30 Cycles: 94°C 15-30 seconds; 45-68°C 15-60 seconds; 68°C 1 minute per kb
Final Extension 68°C 5 minutes
Hold 4-10°C

general guidelines

1.Template:
 Use of high quality, purified DNA templates greatly enhances the success of PCR. Recommended amounts of DNA template of a 50 µL reaction are as follows:

DNA Amount
genomic 1 ng - 1 µg
plasmid or viral 1 pg - 1 ng

2.Primers:
 Oligonucleotide primers are generally 20-40 nucleotides in length and ideally have a GC content of 40-60%. Computer programs such as Primer3 can be used to design or analyze primers. The final concentration of each primer in a PCR may be 0.05-1 µM. typically 0.2 µM.

3.Mg++ and Additives: Mg++ concentration of 1.5-2.0 mM is optimal for most PCR products generated with OneTaq DNA Polymerase. The final Mg++ concentration in 1X OneTaq Quick-Load Master Mix with Standard Buffer is 1.8 mM. This supports satisfactory amplification of most amplicons. Howeoever, Mg++ can be further optimized in 0.2 mM increments using MgCl2.
 For amplification of difficult targets, like GC-rich sequences, we recommend OneTaq Quick Load 2X Master Mix with GC Buffer. Alternatively, DMSO or formamide may be used.

4.Denaturation:
 An initial denaturation of 30 seconds at 94°C is sufficient to amplify most targets from pure DNA templates. For difficult templates such as GC-rich sequences, a longer denaturation of 2-4 minutes at 94°C is recommended prior to PCR cycling to fully denature the template. Alternatively, use OneTaq Quick-Load 2x Master Mix with GC Buffer. With colony PCR, an initial 2-5 minute denaturation at 94°C is recommended to lyse cells.
 During thermocycling a 15-30 second denaturation at 94°C is recommended.

5.Annealing:
 The annealing step is typically 15-60 seconds. Annealing temperature is based on the Tm of the primer pair and is typically 45-68°C. Annealing temperatures can be optimized by doing a temperature gradient PCR starting 5°C below the calculated Tm. We recommend using NEB`s Tm Calculator to determine appropriate annealing temperature for PCR.
 Tm Calculator: http://tmcalculator.neb.com/#!/

6.Extension:
 The recommended extension temperature is 68°C. Extension times are generally 1 minute per kb. A final extension of 5 minutes at 68°C is recommended.

7. Cycle Number:
 Generally, 25-35 cycles yields sufficient product. Up to 45 ycles may be required to detect low copy number targets.

8. 2-step PCR:
 When primers with annealing temperatuers of 68°C or above are used, a 2-step thermocycling potocol (combining annealing and extension into one step) is possible.

9. PCR Product: The majority of the PCR products generated using OneTaq DNA Polymerase contain dA overhangs at the 3`end; therefore the PCR products ca be ligated to dT/dU-overhang vectors.

Materials

 Plate of cells to be made competent
 TSS-medium
 LB Medium
 Ice

Glassware & Equipment

 Falcon tubes
 500μl Eppendorf tubes, on ice
 200ml conical flask
 200μl pipetman or repeating pipettor
 5ml pipette

Preparation

1. Grow a 5ml overnight culture of cells in LB media. In the morning, dilute this culture back into 25-50ml of fresh LB media in a 200ml conical flask. You should aim to dilute the overnight culture by at least 1/100.
2. Grow the diluted culture to an OD600 of 0.2 - 0.5. (You will get a very small pellet if you grow 25ml to OD600 0.2)
3. Put eppendorf tubes on ice now so that they are cold when cells are aliquoted into them later. If your culture is X ml, you will need X tubes. At this point you should also make sure that your TSS is being chilled (it should be stored at 4oC but if you have just made it fresh then put it in an ice bath).
4. Split the culture into two 50ml falcon tubes and incubate on ice for 10 min.

All subsequent steps should be carried out at 4oC and the cells should be kept on ice wherever possible

1. Centrifuge for 10 minutes at 3000 rpm and 4oC.
2. Remove supernatant. The cell pellets should be sufficiently solid that you can just pour off the supernatant if you are careful. Pipette out any remaining media.
3. Resuspend in chilled TSS buffer. The volume of TSS to use is 10% of the culture volume that you spun down. You may need to vortex gently to fully resuspend the culture, keep an eye out for small cell aggregates even after the pellet is completely off the wall.
4. Add 100 μl aliquots to your chilled eppendorfs and store at − 80oC.
5. Place the cells at -80°C

Note:
 If you run a control every time you clone (i.e. a vector-only ligation), you can as well freeze cells in 200 μl aliquots.
 It is a good idea to run a positive control on the cells.

 the instruction for preparing the biobrick: http://parts.igem.org/Help:Protocols/Linearized_Plasmid_Backbones

• 1 Restriction Digest of the gene you want to integrate and pSB1C3
• 2 PCR clean up
• 3 NanoVue
• 4 Dephosphorylation
• 5 Ligation
• 6 Transformation in DH5α

Restriction Digest of the gene you want to integrate and pSB1C3

 We carried out two Double Digests:
 Gen with EcoRI and PstI (we integrated the sequences for this enzymes around our gen before ordering it)
 pSB1C3 linearized plasmid backbone with EcoRI and PstI (this linearized fragment is in the iGEM-Kit)
 digest mix

component backbone pSB1C3 insert
buffer cutsmart (10x) "NBCS" - 1µl - 1µl
EcoRI-HF "N8" - 0,5µl - 0,5µl
PstI-HF "N22" - 0,5µl - 0,5µl
template DNA final c= 10ng/µl - 4µl - ? µl
H20 - 4µl - 6µl

 * amount of DNA:

a) c(0) of backbone: 25ng/µl → to get a final concentration of 10ng/µl we have to use 4 µl backbone-DNA. (because: 25ng/µl * 4µl = 100ng ; 100ng / 10µl = 10ng/µl)
b) c(0) of insert: ___ ng/µl → to get a final concentration of 10ng/µl we have to use ___ µl insert-DNA. (because: __ng/µl * __µl = 100ng ; 100ng / 10µl = 10ng/µl)

 Digest 37°C/30 min, heat kill 80°C/20 min

PCR clean up

 performe a PCR-clean up to remove the enzymes. Use less elution-buffer, because your concentrations will be very low! (Remember: you just use 100ng DNA → the maximal concentration you could get when using 10µl elution buffer is 10ng/µl)

NanoVue

 Performe a NanoVue to determine the concentration of the digested DNA.

Dephosphorylation

It is an optional step which is used when only one restriction enzymes is used. See link: http://igem.rwth-aachen.de/2016/index.php/Protocols/dephosphorylation.

Ligation

 (protocol from iGEM.org)
 ligation mix

digested backbone - 25ng - ___ µl
digested insert - * - µl
T4 DNA ligase buffer - - 1µl
T4 DNA ligase - - 1µl
H20 - - up to 10 µl *

 Ligate 16°C/30 min, heat kill 80°C/20 min
 Transform with 1-2 µl of product

 Note: For linearized plasmid backbones provided by iGEM HQ, a plasmid backbone with an insert of BBa_J04450 was used as template. As a result any red colonies that appear during your ligation may be due to the template as a background. Digesting with Dpn1 before use should reduce this occurrence.

 *calculation of the amount of insert in ng: insert(ng) = plasmid (ng) x [lenght of insert (bp) / lenght of backbone (bp)] x ratio of insert/backbone

 insert ___ (ng) = _ng x [_ bp / _ bp] x 10/1 = ___ ng
 ratio: insert:backbone is 10:1
 concentration of backbone: _ ng/µl ; we want to add 25ng so we have to use 25ng/ __ ng/µl = __ µl
 concentration of insert: _ ng/µl ; we want to add about _ ng so we have to use __ ng/ __ ng/µl = __ µl

Transformation in DH5α

 work space: clean bench

1. Thaw 100 µL E. coli DH5α on ice
2. Add DNA (2µL (~20ng) ligation mixture) to the competent cells and swirl gently
3. Incubate for 15-30 min on ice.
4. Perform heatshock of the competent cells (using a heat-block) at 42°C for 45s (DH5α).
5. Samples were immediately cooled down 2 min on ice.
6. Fill up the transformation mix to 1 mL with LB media or SOC media and incubate at 37°C at 250rpm for 45 min (45-60 min) for recovery of the cells.
7. Plate cells on LB agar plate with antibiotic respectively and dry them under the clean bench.
a. 200 µL
b. resuspended pellet (from centrifugation of the leftover)
8. Incubate the agar plates at 37°C overnight (16-18 hours).
9. Count single colonies of each agar plate on the next day.

Before You Start

Estimated bench time: 1 hour Estimated total time: 2 hours (plus 14-18 hour incubation)

When transforming competent cells, both timing and temperature are very important. Use a lab timer, follow the incubation temperatures closely, and keep materials on ice when required. Read through the entire protocol before starting!

Materials

 Resuspended DNA to be transformed
 10pg/µl Positive transformation control DNA (e.g. pSB1C3 w/ BBa_J04450, RFP on high-copy chloramphenicol resistant plasmid. Located in the *Competent Cell Test Kit.)
 competent cells (50µl per sample)
 1.5mL Microtubes
 SOC Medium (950µL per sample)
 LB Agar + Chloramphenicol(2 per sample.)

Biobricks contain CHL-resistance

Equipment

 Floating Foam Tube Rack
 Ice & ice bucket
 Lab Timer
 42°C water bath
 37°C incubator
 Sterile spreader or glass beads (/ also "dreigalsky-spatel" works)
 Pipettes and Tips (10µl, 20µl, 200µl recommended)
 Microcentrifuge

Method

1. Resuspend DNA in selected wells in the Distribution Kit with 10µl dH20. Pipet up and down several times, let sit for a few minutes. Resuspension will be red from cresol red dye.
2. Label 1.5ml tubes with part name or well location. Fill lab ice bucket with ice, and pre-chill 1.5ml tubes (one tube for each transformation, including your control) in a floating foam tube rack.
3. Thaw competent cells on ice: This may take 10-15min for a 260µl stock. Dispose of unused competent cells. Do not refreeze unused thawed cells, as it will drastically reduce transformation efficiency.
4. Pipette 50µl of competent cells into 1.5ml tube: 50µl in a 1.5ml tube per transformation. Tubes should be labeled, pre-chilled, and in a floating tube rack for support. Keep all tubes on ice. Don’t forget a 1.5ml tube for your control.
5. Pipette 1µl of resuspended DNA into 1.5ml tube: Pipette from well into appropriately labeled tube. Gently pipette up and down a few times. Keep all tubes on ice.
6. Pipette 1µl of control DNA into 2ml tube: Pipette 1µl of 10pg/µl control into your control transformation. Gently pipette up and down a few times. Keep all tubes on ice.
7. Close 1.5ml tubes, incubate on ice for 30min: Tubes may be gently agitated/flicked to mix solution, but return to ice immediately.
8. Heat shock tubes at 42°C for 45 sec: 1.5ml tubes should be in a floating foam tube rack. Place in water bath to ensure the bottoms of the tubes are submerged. Timing is critical.
9. Incubate on ice for 5min: Return transformation tubes to ice bucket.
10. Pipette 950µl SOC media to each transformation: SOC should be stored at 4°C, but can be warmed to room temperature before use. Check for contamination.
11. Incubate at 37°C for 1 hours, shaking at 200-300rpm
12. Pipette 100µL of each transformation onto petri plates Spread with sterilized spreader or glass beads immediately(/"Dreigalsky-Spatel"). This helps ensure that you will be able to pick out a single colony.
13. Spin down cells at 6800g for 3mins and discard 800µL of the supernatant. Resuspend the cells in the remaining 100µL, and pipette each transformation onto petri plates Spread with sterilized spreader or glass beads immediately. This increases the chance of getting colonies from lower concentration DNA samples.
14. Incubate transformations overnight (14-18hr) at 37°C: Incubate the plates upside down (agar side up). If incubated for too long, colonies may overgrow and the antibiotics may start to break down; un-transformed cells will begin to grow.
15. Pick single colonies: Pick single colonies from transformations: do a colony PCR to verify part size, make glycerol stocks, grow up cell cultures and miniprep.
16. Count colonies for control transformation: Count colonies on the 100μl control plate and calculate your competent cell efficiency. Competent cells should have an efficiency of 1.5x10^8 to 6x10^8 cfu/µg DNA.

needed material

 YPD+hygromycin (conc. 250mg/L) plates  YPD plates  auxotrophie-media-plates of the auxotrophie you want to remove  liquid induce-media, with the GAL1-Promotor 100%Galactose YPD-Media  plasmid containing cre- protein and an inducible promoter upstream (e.g. pSH69 of E.coli Stammnummer 2757 (iAMB))

Procedure

1. Transformation of the cells that have to be treated with the plasmid that contains the gene for the cre-protein. For example the plasmid pSH69 from E.coli Stammnummer 2757 (iAMB). The protocol will be described in usage of this plasmid, which contains a GAL1 Promoter upstream of the cre-protein-DNA and a hygromycin Marker Gene. 2. The Transformants get plated on YPD + hygromycin (conc. 250mg/L) plates. 3. When the colonies on the plate grew, use the method "Abschwämmen" to dilute the Colonies on Medium. That means you pour some induce- media onto the plate, so all colonies are covered with media. Than you remove all the colonies from the plate into the media with a drigalski-spatel or something else. 4.Take the media from the plates with all the cells from the colonies in it and pour it into a induce- media, in this case Galactose+YPD -media (because of the GAL1 Promoter). 5.Leave the cells in the induce- media for 4-5h at 28-30°C. Now the cre-protein gets expressed and it deletes all the DNA sequences which are between LOXp- sites. 6.Plate the cells on a YPD- plate but before prepare a dilution-row (Verdünnungsreihe) of the cells in the induce media to get single colonies on the plate. 7.After grwoth of colonies on the YPD-plate, check 5 colonies or more for success of the cre treatment: You mark 5 colonies or more on the YPD- plate and take half of each to spread them on auxotrophie-media-plates of exactly the auxotrophie you want to remove from your cells. Only if the cells can NOT grow on the auxotrophie-media-plate, the cre-treatment was successful!

Materials

 Frozen Competent Cell (FCC) Solution → Solution is 5% (v/v) glycerol and 10% (v/v) DMSO. Be sure to filter-sterilize.
 Liquid YPD media → about 1L
 50 mL Falcon
 styrofoam-box for freezer
 A 250 mL sterile culture flask for each strain
 A 2 L sterile flask for each strain

Cell Preparation

 Day 1
 Inoculate 25ml of YPD with your strain (for an overnight cuture) and grow at 30°C and 200 RPM overnight or for 12-16 hours.
 Day 2
 Pre-warm 500 mL YPD in the 2 L flask at 30°C for later use.
 Take the overnight culture and find the OD600 to determine cell count (OD 0.1 ≈ 1 x 10^6 cells/mL.) An OD close to 1 is desired. (dilute the culture with YPD-medium until you get an OD close to 1)
 With this culture, inoculate the pre-warmed flask of YPD with the entire 25mL and shake at 30°C for about 4 hours.
 NOTE that adding cells according to their titers as described in Geitz et. al will not be possible since the cell densities achieved were not *even close to what was described (try using 2X YPD if this is the route you need to go in.)
 centrifuge at 3,000xg for 5 minutes using 50 mL conical tubes. I split 250 mL of the culture among 5 tubes, spin them down, discard the supernatant, and add the remaining culture to the same tubes and repeat.
 Wash the cells in 0.5 volumes (25 mL in each of these tubes) of sterile water. Resuspend in 0.01 volumes (500μL each) sterile water and transfer to microfuge tubes. Pellet cells at 3,000xg for 5 minutes.
 Resuspend in 500μL FCC solution and aliquot 50μL each into microfuge tubes.
 Place these tubes into the styrofoam racks and store at -80°C. Since the racks are to promote slow freezing, after the tubes are brought to -80°C you may transfer them to whatever your container of choice is in the -80.

Using These Cells for Transformation

 Take out a tube for each set of transformations and warm between your hands for 15 to 30 seconds. Pellet the cells at 3,000xg for 2 minutes and discard the supernatant. Take these cells and add them to your transformation master mix and follow the procedure as described in McClean:Yeast Transformation day 2 step 7. You may need to use more than one tube of cells if your yield was low.

Preparation of Competent Cells
1. Grow yeast cells at 30°C in 10 ml YPD broth until mid-log phase (~5 x 10^6- 2 x 10^7cells/ml or OD600 of 0.8-1.0).

Note
If you don´t know when your mid-log phase is, you can use a fully grown S. cerevisiae overnight culture, dilute it 1/10 and then let it grown for another 4-6 h at 30°C.

The following steps are accomplished at room temperature:

1. Pellet the cells at 500 x g for 4 minutes and discard the supernatant.
2. Add 10 ml EZ 1 solution to wash the pellet. Repellet the cells and discard the supernatant.
3. Add 1 ml EZ 2 solution to resuspend the pellet.

At this point, the competent cells can be used for transformations directly or stored frozen at or below -70°C for future use.

Note:

It is important to freeze the cells slowly. To accomplish this, either wrap the aliquotted cells in 2-6 layers of paper towels or place in a Styrofoam box before placing in the freezer.

DO NOT use liquid nitrogen to snap-freeze the cells.

Transformation

This part of the procedure is the same for both frozen stored (thawed at room temperature) and freshly prepared competent yeast cells.

1. Mix 50 µl of competent cells with 0.2-1 µg DNA (in less than 5 µl volume); add 500 µl EZ 3 solution and mix thoroughly.
2. Incubate at 30°C for 45 minutes. Mix vigorously by flicking with finger or vortexing (if appropriate for your DNA) 2-3 times during this incubation.
3. Spread 50-150 µl of the above transformation mixture on an appropriate plate. It is unnecessary to pellet and wash the cells before spreading.
4. Incubate the plates at 30°C for 2-4 days to allow for growth of transformants.

Attention:

For transformations of C. albicans, use freshly prepared competent cells; frozen cells sometimes give poor results.

[URA-]-medium with synthetic drop-out complement

Composition:

 960ml ddH2O
 6.7g nitrogen base without AA
 40ml 50% glucose solution
 1.92g synthetic drop-out complement ([URA-])

Note:
Glucose should only be sterile filtered added to the mixture. It should be done after autoclaving the mixture. Glucose should not be autoclaved because of the possible caramelisation of it due to the high temperature. Storage at room temperature.

[URA-]-plates with synthetic drop-out complement

Composition:

 960ml ddH2O
 6.7g nitrogen base without AA
 40ml 50% glucose solution
 1.92g synthetic drop-out complement ([URA-])
 20g Agarose

Note:
Glucose should only be sterile filtered added to the mixture. It should be done after autoclaving the mixture. Glucose should not be autoclaved because of the possible caramelisation of it due to the high temperature. storage at 4°C.

[HIS-]-medium with synthetic drop-out complement

Composition:

 960ml ddH2O
 6.7g nitrogen base without AA
 40ml 50% glucose solution
 1.92g synthetic drop-out complement ([URA-])

alternative to 1.92g synthetic drop-out complement ([HIS-]:
50ul of 1% uracyl, thryptophan and leucin + synthetic drop-out complement ([HIS-],[URA-],[TRP-] and [LEU-])

Note:
Glucose should only be sterile filtered added to the mixture. It should be done after autoclaving the mixture. Glucose should not be autoclaved because of the possible caramelisation of it due to the high temperature. Storage at room temperature.

[URA-]-plates with synthetic drop-out complement

Composition:

 960ml ddH2O
 6.7g nitrogen base without AA
 40ml 50% glucose solution
 1.92g synthetic drop-out complement ([URA-])

alternative to 1.92g synthetic drop-out complement ([HIS-]: 50ul of 1% uracyl, thryptophan and leucin + synthetic drop-out complement ([HIS-],[URA-],[TRP-] and [LEU-])

 20g Agarose

Note: Glucose should only be sterile filtered added to the mixture. It should be done after autoclaving the mixture. Glucose should not be autoclaved because of the possible caramelisation of it due to the high temperature. storage at 4°C.

all mentioned synthetic drop-out complements are avaible on the internet. We used syntheticdrop-out complements of sigma-aldrich, wich are in Schwaneberg laboratory avaible