Inverse PCR

For use with Snyder mTn-lacZ/LEU2 based mutagenesis

1. Genomic DNA Prep
from 5 ml culture, resuspend in 50 µl TE


2. Digestions

Genomic DNA	5 µl
10x NEB Buffer	5 µl
0.5 µg/µl RNaseA	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


3. 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 NH₄Ac + ethanol…-20 deg C/ >1 hr; spin, dry, resuspend pellet in 100 µl TE


4. 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
H2O	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'



5. Cleanup for Sequencing (2 options)
1. Wizard PCR purification spin columns
elute in 50 ul TE
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


6. Sequencing

Cleaned DNA	10.5 µl
Sequencing Mix	8 µl
DMSO	1 µl
Sequencing Oligo, 10 µM	0.5 l

• 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'