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Common Laboratory Protocols
- Bacterial Transformation
- Colony PCR
- Gel Extraction
- Ligation
- Miniprep
- PCR Purification
- PCR Reaction
- Restriction Enzyme Digest
- TdT Heat Inactivationn
- TdT Nucleotide Addition
- Yeast Gene Knockout
- Yeast Mating
- Yeast Sporulation/Zymolyase Treatment
Bacterial Transformation
- Materials
- plasmid DNA (up to 100ng, no more than 5μL)
- Competent bacteria aliquot (stored -80°C)
- SOC (stored 4°C)
- Prewarm 2 water baths to 37°C and 42°C
- Aliquot 100μL SOC into microcentrifuge tube. Prewarm in 37°C.
- Prewarm LB plate + resistance to 37°C.
- Add DNA to compontent cells. Incubate on ice 15-20 min.
- Heat shock by quickly transferring tubes from ice to 42°C water bath for EXACTLY 45 seconds.
- Recuperate for 2 minutes on ice.
- Transfer prewarmed SOC to bacteria. Incubate 30 min 37°C.
- Transfer all the solution to LB plate. Spread and wait 15 min on benchtop.
- Invert and incubate 37°C overnight.
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Colony PCR
Gel Extraction
Miniprep
PCR Purification
PCR Reaction
Restriction Enzyme Digest
- Materials
- 10x Buffer (Compatible to the enzyme)
- Enzyme
- BSA - diluted to 10x
- ddH2O
- DNA - up to 1μg per reaction.
Procedure Verify all times and temperatures for specific enzyme prior to use. This is general protocol.
- Prewarm 2 water baths to 37°C and 70°C
- Thaw buffers, diluted BSA. Keep all reagents on ice
- Set up reactions based on table below.
Reagent Volume Example Reaction Buffer 1/10 total volume 2μL Diluted BSA (10x) 1/10 total volume 2μL DNA calculate for target ng 3.7μL ddH2O volume up to (total volume - 1μL) 11.3μL Enzyme generally 1μL 1μL Total Volume 20μL - Incubate 60min 37°C.
- Heat inactive 70°C for 15 min.
- Store in -20°C or use immediately.
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TdT Heat Inactivation
TdT Nucleotide Addition
Procedure Experiment (repeat for each nucleotide) 1. Pre-heat water baths to 37°C and 95°C 2. Mix: 5.0 uL 10X TdT Buffer 5.0 uL 2.5 mM CoCl2 solution 1ug 43 mer oligo (1.3uL of 813 ng/uL) 0.4 uL 50mM CleanAmp dATP 20 Units TdT (1 uL of 20,000 U/uL) dH2O to final volume of 50 uL 3. Incubate at 37° for 30 minutes. 4. Prepare 2 equal volume aliquots of the mixture (25 uL each). 5. Incubate 1 aliquot at 95° for 15 minutes. 6. Add additional 10 U TdT (0.5 uL of 20,000 U/uL) to both aliqouts. 7. Cool 95°C water bath to 70°C. 8. Incubate at 37° for 60 minutes. 9. Heat inactivate at 70° for 10 minutes. 10. Store at -20°C. Controls: 1)Negative (No TdT) 1. Pre-heat water baths to 37°C and 70°C 2. Mix: 5.0 uL 10X TdT Buffer 5.0 uL 2.5 mM CoCl2 solution 500 ng 24-mer oligo (0.9 uL of 568.8 ng/uL) 1.0 uL 10mM dATP dH2O to final volume of 50 uL 3. Incubate at 37° for 60 minutes 9. Heat inactivate at 70° for 10 minutes. 10. Store at -20°C. 2)Five Minute Incubation Time 1. Pre-heat water baths to 37°C and 70°C 2. Mix: 5.0 uL 10X TdT Buffer 5.0 uL 2.5 mM CoCl2 solution 500 ng 24-mer oligo (0.9 uL of 568.8 ng/uL) 1.0 uL 10mM dATP 10 Units TdT (0.5 uL of 20,000 U/uL) dH2O to final volume of 50 uL 3. Incubate at 37° for 5 minutes 9. Heat inactivate at 70° for 10 minutes. 10. Store at -20°C. 3)Sixty Minute Incubation Time 1. Pre-heat water baths to 37°C and 70°C 2. Mix: 5.0 uL 10X TdT Buffer 5.0 uL 2.5 mM CoCl2 solution 500 ng 24-mer oligo (0.9 uL of 568.8 ng/uL) 1.0 uL 10mM dATP 10 Units TdT (0.5 uL of 20,000 U/uL) dH2O to final volume of 50 uL 3. Incubate at 37° for 60 minutes 9. Heat inactivate at 70° for 10 minutes. 10. Store at -20°C.
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