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#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 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

 

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'