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

Qiagen protocol generally used

Materials

DNA sample ran through agarose gel

sharp edge, like scalpel or gel cutter

Qiagen Buffers QG, PE, EB

Isopropanol

Procedure

1. Prewarm waterbath to 50°C
2. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
3. Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg ~ 100 µl)
4. Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). To help dissolve gel, mix by vortexing the tube every 2–3 min during the incubation.
5. After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose).
6. Add 1 gel volume of isopropanol to the sample and mix.
7. Place a QIAquick spin column in a provided 2 ml collection tube.
8. To bind DNA, apply the sample to the QIAquick column, and centrifuge for 1 min.
9. Discard flow-through and place QIAquick column back in the same collection tube.
10. (Optional): Add 0.5 ml of Buffer QG to QIAquick column and centrifuge for 1 min.
11. To wash, add 0.75 ml of Buffer PE to QIAquick column and centrifuge for 1 min.
12. Discard the flow-through and centrifuge the QIAquick column for an additional 1 min at ≥10,000 x g (~13,000 rpm).
13. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.
14. To elute DNA, add 50 µl of Buffer EB (10 mM Tris·Cl, pH 8.5) or H2O to the center of the QIAquick membrane and centrifuge the column for 1 min at maximum speed.

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Miniprep

Qiagen protocol generally used

Materials

5mL of overnight bacteria culture

Qiagen Buffers P1, P2, N3, PE, EB

Procedure

1. Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to a microcentrifuge tube.
2. Add 250 µl Buffer P2 and gently invert the tube 4–6 times to mix
3. Add 350 µl Buffer N3 and invert the tube immediately but gently 4–6 times.
4. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
5. Apply the supernatants from step 4 to the QIAprep spin column by decanting or pipetting.
6. Centrifuge for 30–60 s. Discard the flow-through.
7. (Optional): Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30–60 s. Discard the flow-through.
8. Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30–60 s.
9. Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer.
10. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 µl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.

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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

Materials

DNA source - commercially made oligo's

Terminal Transferase (NEB - M0315S)

Procedure

• Pre-heat water baths to 37°C, 75°C, and 95°C
• Mix 8 samples on ice:

  1.0 μL 10X TdT Buffer

  1.0 μL 2.5 mM CoCl₂

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

  1.0 μL 10mM dNTP

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

  dH₂O to final volume of 10 μL

Incubate at 95°C for 0, 0.5, 1, 2, 5, 10, 15, and 20 minutes.

Incubate at 37°C for 10 minutes.

Heat inactivate at 75° for 20 minutes.

Store at -20°C.

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TdT Nucleotide Addition

Materials

DNA source - commercially made oligo's

Terminal Transferase (NEB - M0315S)

CleanAmp dNTP's (TriLink - N9507)

Procedure
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