Inverse PCR For use with Snyder mTn-lacZ/LEU2 based mutagenesis
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Inverse PCR
I. Genomic DNA Prep
• from 5 ml culture, resuspend in 50 µl TE
II. Digestions
genomic DNA |
5 µl |
10x of appropriate NEB buffer |
5 µl |
0.5 µg/µl RNase |
1 µl |
H2O |
38 µl |
restriction enzyme |
1 µl |
• Use either AciI, AluI, HaeIII, HpaII, RsaI or TaqI for Leu transposon libraries
• 37 deg C/ >3 hours (or overnight)
• 65 deg C/ 20 min
III. Ligations (intramolecular, hopefully)
digested DNA |
10 µl |
10x ligation buffer |
20 µl |
H2O |
~170 µl |
T4 DNA ligase (NEB, 400U/µl) |
0.2 µl |
• all day at room temp or overnight at 4 deg C
• precipitate with 80 µl 5M NH4 Ac + ethanol…-20 deg C/ >1 hr; spin, dry, resuspend pellet in 100 µl TE
IV. PCR
ligated DNA |
10 µl |
3 M KCl |
0.75 µl |
1 M Tris, pH 8.5 |
0.4 µl |
25 mM MgCl2 |
3 µl |
10 mM dNTPs |
1 µl |
10 µM oligo#undefined |
1 µl |
10 µM oligo#undefined |
1 µl |
H2 O |
32.35 µl |
Taq (added after hot start) |
1 µl |
• 35 cycles of: 94 deg C/1', 62 deg C/1', 72 deg C/2'30"
Enzyme |
Oligos for PCR |
AciI |
InPCR3 and InPCR4 |
AluI |
InPCR3 and InPCR4 |
HaeIII |
InPCR3 and InPCR4 |
HpaII |
InPCR3 and InPCR4 |
RsaI |
InPCR1 and InPCR2 or |
TaqI |
InPCR1 and InPCR2 or |
InPCR2 => 5'-ttccatgttgccactcgctttaatg-3'
InPCR3 => 5'-ataactacgatacgggagggcttacc-3'
InPCR4 => 5'-gattaagcattggtaactgtcagacc-3'
InPCR5 => 5'-cataattctcttactgtcatgccatcc-3'
InPCR6 => 5'-tcaaggatcttaccgctgttgagatcc-3'
V. Cleanup for Sequencing (2 options)
1. Wizard PCR purification spin columns
• elute in 50 µl TE
OR:
2. Exonuclease I+ shrimp alkaline phosphatase treatment:
• In PCR tubes, add:
1 µl Exonuclease I
1 µl SAP (shrimp alkaline phosphatase)
• 37 deg C/20 min
• 65 deg C/20 min
VI. Sequencing
cleaned DNA |
10.5 µl |
sequencing mix |
8 µl |
DMSO |
1 µl |
sequencing oligo �10 µM |
0.5µl |
• use Amersham cycle seq protocol (96 deg C/30", 45 deg C/15", 60 deg C/4'; 30 cycles for dilute templates)
• after cycling, put rxn through a Pharmacia Auto-Seq G-50 column, and dry
• if using the Exo/SAP treatment above, then just use the entire reaction for sequencing
• for sequencing from InPCR1-2 products, use mTn3-SEQ1 oligo
mTn3-SEQ1 => 5'-cccccttaacgtgagttttcgttccact-3'• for sequencing from InPCR3-4, InPCR4-5, or InPCR4-6 products use mTn3-SEQ2 oligo
mTn3-SEQ2 => 5'-aaggatctaggtgaagatcc-3'