Common Laboratory Protocols

• Bacterial Transformation
• Colony PCR
• Gel Extraction
• 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)

1. Prewarm 2 water baths to 37°C and 42°C
2. Aliquot 100μL SOC into microcentrifuge tube. Prewarm in 37°C.
3. Prewarm LB plate + resistance to 37°C.
4. Add DNA to compontent cells. Incubate on ice 15-20 min.
5. Heat shock by quickly transferring tubes from ice to 42°C water bath for EXACTLY 45 seconds.
6. Recuperate for 2 minutes on ice.
7. Transfer prewarmed SOC to bacteria. Incubate 30 min 37°C.
8. Transfer all the solution to LB plate. Spread and wait 15 min on benchtop.
9. Invert and incubate 37°C overnight.

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.

1. Prewarm 2 water baths to 37°C and 70°C
2. Thaw buffers, diluted BSA. Keep all reagents on ice
3. 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

4. Incubate 60min 37°C.
5. Heat inactive 70°C for 15 min.
6. Store in -20°C or use immediately.

TdT Heat Inactivation

TdT Nucleotide Addition

Yeast Gene Knockout

Yeast Mating

Yeast Sporulation/Zymolyase Treatment