Difference between revisions of "PhiX:Mutagenesis"

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COMPARE to my protocol
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'''This protocol is now superseded - UPDATE SOON'''<br>
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''With credit to Bentley Fane and Maniartis.''
  
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Primer phosphorylation with T4 PNK: we phosphorylize 200 pmol of primer in 2ul reaction mixture
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==Step 1: Primer phosphorylation==
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We phosphorylate 200pmol primer (Fane carries this out in 2ul reaction mixture):
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* 2ul 100uM primer
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* 2ul 10mM ATP
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* 2ul T4 ligase buffer
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* 1ul 10U/ul T4 polynucleotide kinase
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* 13ul water
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-> 37<sup>o</sup>C for 30 minutes.
  
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Annealing: 10 pmol primer, 0.5 p mole ssDNA in 10ul PE1 buffer (10XPE1: 200mM Tris ph &.5; 100mM MgCl2, 200 mM NaCl, 10 mM DTT).
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==Step 2: Relax secondary structure and anneal==
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We add 10pmol of phosphorylated primer to 0.5pmol ssDNA in ligase buffer (Fane uses [[PhiX:Recipes|PE1 buffer]]). Start boiling water in a bowl using tripod arrangement. Meanwhile to each tube add:
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* 2ul ssDNA diluted 1/100, 1/10, or 1 times (SHOULD PURIFY BAND)
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* 1ul phosphorylated primer mix (above)
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* 6ul water
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* 1ul T4 ligase buffer
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When water is boiled, remove bowl from heat and immerse tubes in it. Store bowl (with tubes) in cold room or fridge (+4<sup>o</sup>C) for 40 minutes to 1 hour.
  
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phiX174 DNA has a great deal of secondary structure. To anneal, we heat some
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==Step 3: Non-gapped extension reaction==
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water to boiling, remove from heat, place the tube in the water, refrigerate
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We add ATP to 1mM, dNTPs to 0.5mM, 4 Weiss Units ligase, 2U polymerase in ligase buffer to 20ul (Fane uses [[PhiX:Recipes|PE1 buffer]]). The following is added to each tube (error here unless 10ul in previous):
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and let it cool to 4C, takes about an hour or two.
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* 1ul ligase buffer
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* 2ul 10mM ATP
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* 1ul 2mM (each) dNTPs
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* 3.3ul water
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* 0.7ul 3U/ul T4 DNA polymerase
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* 2ul 2U/ul (400 c.e.u.s) T4 DNA ligase
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Reaction kept on ice for 5 minutes, room temperature for 5 minutes, then two hours at 37<sup>o</sup>C. After which the reaction may be frozen.
  
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We then add ATP to 1 mM, nucelotides to 0.5 mM, four units of T4 ligase, and 2
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==Step 4: Prepare for transformation==
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units T4 DNA polymerase. This reaction is done in PE2 buffer (10X PE2 200 mM
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Fane uses 2ul of 1/10 dilution of this for transformation into 100ul competent cells. Transformation efficiency improved by O/N storage.
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Tris Ph 7.5, 100 mM MgCl<sub>2</sub>, 100 mM DTT). Total volume of the reaction is 20
 
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micro liters.
 
  
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Keep on ice for 5 minutes, then room temp for five minutes, then 2
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From Fane:
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hours at 37C.
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We incubate the transformation mixtures for 30 minutes on ice, heat shock for 1 minute at 42<sup>o</sup>C, then ice again for 5 minutes, and then we plate. This
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After wards the reactiosn can be frozen. We typically dilute the reaction 1/10
 
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before transformation. The transformation mixture contains 100 microliter
 
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competent cells, and we use 2 microliter of the diluted synthesis reaction.
 
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We have found that CaCl2 treatement gives us the best transformations. We also
 
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get much better efficiencies if we make the competent cells the day
 
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before, and
 
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refrigerate them overnight.
 
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We incubate the transformation mixtures for 30 minutes on ice, heat shock for
 
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one minute at 42, then ice again for 5 minutes, and then we plate. This
 
 
typically gives us 200 plaques per plate.
 
typically gives us 200 plaques per plate.
  
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The repair can go either way in the cells, so you will need a way to either
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The repair can go either way in the cells, so you will need a way to either directly select for your mutant, or an efficient way to screen the plaques by toothpicking into indicator lawns.
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directly select for your mutant, or an efficient way to screen the plaques by
 
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toothpicking into indicator lawns.
 

Latest revision as of 19:19, 6 August 2013

This protocol is now superseded - UPDATE SOON
With credit to Bentley Fane and Maniartis.

Step 1: Primer phosphorylation

We phosphorylate 200pmol primer (Fane carries this out in 2ul reaction mixture):

  • 2ul 100uM primer
  • 2ul 10mM ATP
  • 2ul T4 ligase buffer
  • 1ul 10U/ul T4 polynucleotide kinase
  • 13ul water

-> 37oC for 30 minutes.

Step 2: Relax secondary structure and anneal

We add 10pmol of phosphorylated primer to 0.5pmol ssDNA in ligase buffer (Fane uses PE1 buffer). Start boiling water in a bowl using tripod arrangement. Meanwhile to each tube add:

  • 2ul ssDNA diluted 1/100, 1/10, or 1 times (SHOULD PURIFY BAND)
  • 1ul phosphorylated primer mix (above)
  • 6ul water
  • 1ul T4 ligase buffer

When water is boiled, remove bowl from heat and immerse tubes in it. Store bowl (with tubes) in cold room or fridge (+4oC) for 40 minutes to 1 hour.

Step 3: Non-gapped extension reaction

We add ATP to 1mM, dNTPs to 0.5mM, 4 Weiss Units ligase, 2U polymerase in ligase buffer to 20ul (Fane uses PE1 buffer). The following is added to each tube (error here unless 10ul in previous):

  • 1ul ligase buffer
  • 2ul 10mM ATP
  • 1ul 2mM (each) dNTPs
  • 3.3ul water
  • 0.7ul 3U/ul T4 DNA polymerase
  • 2ul 2U/ul (400 c.e.u.s) T4 DNA ligase

Reaction kept on ice for 5 minutes, room temperature for 5 minutes, then two hours at 37oC. After which the reaction may be frozen.

Step 4: Prepare for transformation

Fane uses 2ul of 1/10 dilution of this for transformation into 100ul competent cells. Transformation efficiency improved by O/N storage.

From Fane: We incubate the transformation mixtures for 30 minutes on ice, heat shock for 1 minute at 42oC, then ice again for 5 minutes, and then we plate. This typically gives us 200 plaques per plate.

The repair can go either way in the cells, so you will need a way to either directly select for your mutant, or an efficient way to screen the plaques by toothpicking into indicator lawns.

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