Inverse PCR For use with Snyder mTn-lacZ/LEU2 based mutagenesis

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#1*	1 µl
10 µM oligo#2*	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"

*the choice of oligos used for the PCR reaction depends on what restriction enzyme was initially chosen to cut the genomic DNA and what "side" of the transposon you want to use as the starting point for the PCR.

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 InPCR4 and InPCR5
TaqI	InPCR1 and InPCR2 or InPCR4 and InPCR6

InPCR1 => 5'-taagttgggtaacgccagggttttc-3' 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:

8.5 µl PCR product

1 µl Exonuclease I

1 µl SAP (shrimp alkaline phosphatase)

• 37 deg C/20 min

• 65 deg C/20 min

VI. Sequencing