Difference between revisions of "PhiX:Mutagenesis"

Revision as of 15:47, 24 August 2009

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

-> 37^oC 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 beaker 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 beaker from heat and immerse tubes in it. Store beaker (with tubes) in cold room (+4^oC) for one/two hours.

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 37^oC. 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 42^oC, 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.