Common Laboratory Protocols

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• 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

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Bacterial Transformation

Materials

plasmid DNA (up to 100ng, no more than 5μL)

Competent bacteria aliquot (stored -80°C)

SOC (stored 4°C)

Procedure

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.

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Colony PCR

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Gel Extraction

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Miniprep

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PCR Purification

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PCR Reaction

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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.

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.

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TdT Heat Inactivation

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TdT Nucleotide Addition

repeat for each nucleotide

1. Pre-heat water baths to 37°C and 95°C
2. Mix:

   5.0 μL 10X TdT Buffer

   5.0 μL 2.5 mM CoCl₂ solution

   1μg 43 mer oligo (1.3μL of 813 ng/μL)

   0.4 μL 50mM CleanAmp dATP

   20 Units TdT (1 μL of 20,000 U/μL)

   dH₂O to final volume of 50 μL

3. Incubate at 37°C for 30 minutes.
4. Prepare 2 equal volume aliquots of the mixture (25 μL each).
5. Incubate 1 aliquot at 95° for 15 minutes.
6. Add additional 10 U TdT (0.5 μL of 20,000 U/μL) to both aliqouts.
7. Cool 95°C water bath to 70°C.
8. Incubate at 37μC for 60 minutes.
9. Heat inactivate at 70μC 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 μL 10X TdT Buffer

      5.0 μL 2.5 mM CoCl₂ solution

      500 ng 24-mer oligo (0.9 μL of 568.8 ng/μL)

      1.0 μL 10mM dATP

      dH₂O to final volume of 50 μL

   3. Incubate at 37°C for 60 minutes
   4. Heat inactivate at 70° for 10 minutes.
   5. Store at -20°C.
2. Five Minute Incubation Time
   1. Pre-heat water baths to 37°C and 70°C
   2. Mix:

      5.0 μL 10X TdT Buffer

      5.0 μL 2.5 mM CoCl₂ solution

      500 ng 24-mer oligo (0.9 μL of 568.8 ng/μL)

      1.0 μL 10mM dATP

      10 Units TdT (0.5 μL of 20,000 U/μL)

      dH₂O to final volume of 50 μL

   3. Incubate at 37°C for 5 minutes
   4. Heat inactivate at 70° for 10 minutes.
   5. Store at -20°C.
3. Sixty Minute Incubation Time
   1. Pre-heat water baths to 37°C and 70°C
   2. Mix:

      5.0 μL 10X TdT Buffer

      5.0 μL 2.5 mM CoCl₂ solution

      500 ng 24-mer oligo (0.9 μL of 568.8 ng/μL)

      1.0 μL 10mM dATP

      10 Units TdT (0.5 μL of 20,000 U/μL)

      dH₂O to final volume of 50 μL

   3. Incubate at 37°C for 60 minutes
   4. Heat inactivate at 70° for 10 minutes.
   5. Store at -20°C.

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Yeast Gene Knockout

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Yeast Mating

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Yeast Sporulation/Zymolyase Treatment