PROTOCOLS
Medium
LB Materials
| 10g/L tryptone |
| 5g/L yeast extract |
| 10g/L NaCl |
| (20g/L Agar) |
YPDA Materials
| Bacto petone | 20g/L |
| Yeast extract | 10g/L |
| Glucose | 20g/L |
| (20g/L Agar) |
Minimal SD Base Materials
| Minimal SD Base | 26.7g/L |
| Class defective medium | 1.3g/L DO |
| (20g/L Agar) |
Molecular biology
TIANprep Mini plasmid Kit Materials
| Spin Columns CP3 |
| Collection Tubes 2ml |
| Buffer P1 |
| Buffer P2 |
| Buffer P3 |
| Buffer PW |
| Buffer EB |
Protocols
1. Take 1~5ml of overnight culture E.coli solution, centrifuge at 12000rpm for 1min,then remove the supernatant
2.Resuspend cell precipitation
Add 250ml Buffer P1
Add 250ml Buffer P2 and turn up and down 6~8 times
Add 350ml Buffer P3 and turn up and down 6~8 times, centrifuge at 12000rpm for 10min
Transfer the supernatant to Spin Columns CP3, centrifuge at 12000rpm for 30~60s, pour the waste solution, add 600ml Buffer PW, centrifuge at 12000rpm for 2min, repeat twice.
3. Centrifuge the empty tube at 12000rpm for 2min
Transfer Spin Columns CP3 to a new centrifugal tube, add Buffer EB, and centrifuge at 12000rpm for 2min.
MolPure Gel Extraction Kit Materials
| MolPure DNA Column G1 |
| 2ml collection Tube G1 |
| AC Buffer G1 |
| BD Buffer G1 |
| Wash Buffer |
| Elution Buffer |
| Benchtop centrifuge |
Protocols
1. Sample pretreatment
Weigh the agarose gel containing the target fragment
For every 100mg of 1% agarose gel, 6 times the volume of BD Buffer G1 should be added
50~60℃ water bath for 10min
2. DNA product purification
Add the sample mixture to MolPure DNA Column G1, leave it at room temperature for 1min, centrifuge at 12000rpm for 1min, discard the waste liquid.
Add 700ml Wash Buffer, centrifuge at 12000rpm for 30s, discard the waste solution.
Add 500ml Wash Buffer, centrifuge at 12000rpm for 30s, discard the waste liquid.
Centrifuge the empty tube for 2min, dry at room temperature for 5min
Add 50ml Elution Buffer, centrifuge at 12000rpm for 1min
50μl PCR system
| component | volume |
|---|---|
| dd H2O | 35.5μl |
| 10xtag buffer(Mg2+free) | 5μl |
| 25mM MgCl2 | 3μl |
| Primer -F | 2μl |
| Primer -R | 2μl |
| dNTP MIX | 1μl |
| Template DNA | 1μl |
| Hieff Taq DNA polymerase | 0.4μl |
Transform E.coli Materials
| dh5a |
| LB mediem |
| water bath |
| constant temperature shake |
| Clean bench |
Protocols
1. Thaw a tube Of Competent cells on ice until the last crystals disappear(about 10 min). Mix gently and carefully pipette 50 cells into a transformation tube on ice.
2. Add 1 -5 ul containing 1pg—100ng of plasmid DNA to the cell mixture.Carefully flick the tube 4-5 times t0 mix cells and DNA.DO not vortex.
3. Place the mixture on ice for 30 minutes.Do not mix.
4. Heat shock at exactly 42 ℃ for exactly 42seconds. DO not mix.
5. Place on ice for 3 minutes. Do not mix.
6. Pipette 800ul room temperature LB into the mixture.
7. Place at 37 ℃ for 60 minutes.Shake vigorously （ 180 rpm) or rotate.
8. Warm selection plates to37 ℃
9. Mix the cells thoroughly by flicking the tube and inverting, then centrifuge at 4000 rpm for 1 min,then discard the supernatant to keep 300ul in the tube.
10. Spread 100ul onto a selection plate and incubate overnight at 37 ℃
Infusion connection Materials
| urified PCR fragment |
| Linearized vector |
| 5X In-Fusion HD Enzyme Premix |
| ddH20 |
| water bath |
Protocols
1. Purified PCR fragment 10-200 ng
2. Linearized vector 50-200 ng
3. 5X In-Fusion HD Enzyme Premix 2 μl
4. Deionized water to 10 μl
5. Incubate the reaction system at 50 °C for 15 min and then place on ice
Yeast protocol
yeast double hybridization
Culture medium configuration
YPDA Liquid culture medium，(121°C，Germicidal 15min)
| Tryptone | 2g |
| Yeast Extract | 1g |
| Ade | 0.05g |
| Glucose | 2g |
| Total system | 100ml |
Sterilization in autoclave at 120 degrees for 15 minutes
Preparation of reagents
| 10 xTE | 10xLiAc | 50%PEG | ddH2O | ||
| 1xTE/LiAC | 1.50ml | 150ul | 150ul | 1.20ml | |
| 1xPEG/LiAC | 10ml | 1.0ml | 1.0ml | 8.0ml | |
| 1xTE | 10ml | 1.0ml | 9.0ml |
Master batch preparation:
| 0.5M EDTA | 10ml 1.8612g | EDTA | NaOH--PH=8.0 |
| 1M Tris-HCL | 50ml 6.057g | Tris | HCl --PH=7.5 |
| 10xLiAC | 10ml 1.02g | LiAC | 乙酸--PH=7.5 |
| 50%PEG | 50ml 25g | PEG3350 | |
| 80%Bacteria-preserving glycerin | 80ml |
10×TE: 10 mL of 1M Tris-HCL (pH 7.5), 2 mL of 0.5M EDTA (pH 8.0), add deionized water to 100 mL and adjust the pH to 7.5 with hydrochloric acid.
0.2% Adenine: 0.2g Adenine Hemisulfate Salt dissolved in 100ml ddH2O;
X-α-Gal: 20mg/ml dissolved in DMF (Dimethyl formamide), stored at -20℃ and protected from light.
3-AT (5M): 4.202g 3-AT dissolved in 10ml ddH2O, dissolved in a warm water bath (<50℃), filtered through a sterile filter and divided into 10 EP tubes, stored at - 20℃.
Perform
1. Pick a three-day-old, 2-3 mm diameter AH109 monoclonal from YPDA plate, insert into 1 ml YPDA liquid medium, shake to break up the colony, then insert into 50 ml YPDA liquid medium, incubate overnight (16-18hr) at 30°C with 250rpm incubation, until OD600>1.5;
2. Take appropriate amount of E.coli solution to inoculate to 150 ml fresh YPDA medium to OD600=0.2-0.3, incubate at 30°C with constant temperature and 250rpm until OD600=0.4-0.6 (about 3h);
3. Centrifuge at 700g for 5min at room temperature and discard the supernatant; add 50 ml ddH2O or TE to wash the yeast precipitated cells, centrifuge and discard the supernatant, repeat washing once again, and resuspend the precipitated cells with 1.5 ml 1xTE/LiAc, then get the yeast receptor cells;
4. Dispense the plasmid DNA to be transformed：
| Experimental group | Positive control | Negative control | Dosage |
| pGBKT7-bait | pGBKT7-53 | pGBKT7-lam | 0.2ug |
| pGAT7-Y | pGADT7-T | pGADT7-T | 0.1ug |
| SpermDNA*(10 mg/ml) | 10ul |
6. Add 100ul of yeast receptor cells resuspended with 1xTE/LiAc to each tube, shake and mix well;
7. Add 600ul of PEG/LiAc to each tube, shake vigorously (to improve transformation efficiency), incubate at 30°C with constant temperature and 200rpm for 30min;
8. Add 70ul of DMSO each, mix by slow inversion (no shaking violently), heat shock in water bath at 42°C for 15min, then quickly cool in ice bath for 1-2min;
9. Centrifuge at 6000 rpm for 30sec at room temperature, discard the supernatant as much as possible, take 200ul of 1xTE from each tube, resuspend the precipitated cells, coat SD/ -Trp-Leu and SD/ -Trp-Leu-His solid culture plates, and incubate at 30°C for 3 days in inverted position;
10. Pick colonies and spot seed them on SD/-Trp-Leu-His/X-α-GAL and SD/-Trp-Leu-His-Ade/X-α-GAL plates and incubate at 30°C for 1-2 days.
Day-1
1、Pick 2 BD-bait protein monoclones with an inoculation loop into a 250ml triangular flask containing 50ml SD/-Trp and shake at 250rpm/30°C overnight until OD600=0.8. Note: The monoclones are 2-3mm in diameter.
Day-2
1、Divide the yeast solution into two 50ml centrifuge tubes, centrifuged at 700g/5min and discard the supernatant.
2、Suspend the yeast cells with 2ml 2xYPDA respectively and then combined into one tube.
3、Add the AD library yeast which are melted on ice to the above 4ml yeast solution, repeatedly aspirate and mix.
4、Use 1ml 2xYPDA to repeatedly aspirate the library yeast tube to the above yeast solution, repeat twice.
5、Transfer the mixture into 2L triangle flask, and wash the EP tube into 2L triangle flask with 22ml 2xYPDA twice, incubate at 45 rpm/30℃ for 20-24h.
Day-3
1、take 5ul of yeast solution to microscopic examination, until the yeast solution contains a large number of 结合子.
2、Divide yeast solution into two 50ml centrifuge tubes, 50ml 0.9% NaCl rinse triangular bottles and into the above centrifuge tubes, centrifuge and discard the supernatant.
3、Wash and resuspent the yeast twice with ddH2O and discard the supernatant.
4、resuspend with 7ml 0.9% Nacl, wash the yeast solution to a tube, every 200ul coated with a three amino acids lacking plate.
5、Incubate at 30 ℃ for 3-7 days.
6、monoclonal spot seeding three amino acids lacking color plate, reconfirm.
Yeast pcr system:
1、Pick a small amount of yeast to 15ul 0.2M/NaoH and break the cell wall at 99℃/10min.
2、Take 1.5ul of supernatant after wall breaking for Pcr.
