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Inverse PCR

For use with Snyder mTn-lacZ/LEU2 based mutagenesis


I. Genomic DNA Prep

• from 5 ml culture, resuspend in 50 Ál TE

II. Digestions

genomic DNA

5 Ál

10x of appropriate NEB buffer

5 Ál

0.5 Ág/Ál RNase

1 Ál

H2O

38 Ál

restriction enzyme

1 Ál

• 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 Ál

10x ligation buffer

20 Ál

H2O

~170 Ál

T4 DNA ligase (NEB, 400U/Ál)

0.2 Ál

• all day at room temp or overnight at 4 deg C

• precipitate with 80 Ál 5M NH4Ac + ethanol…-20 deg C/ >1 hr; spin, dry, resuspend pellet in 100 Ál TE

IV. PCR

ligated DNA

10 Ál

3 M KCl

0.75 Ál

1 M Tris, pH 8.5

0.4 Ál

25 mM MgCl2

3 Ál

10 mM dNTPs

1 Ál

10 ÁM oligo#1*

1 Ál

10 ÁM oligo#2*

1 Ál

H2O

32.35 Ál

Taq (added after hot start)

1 Ál

• 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 Ál TE

OR:

2. Exonuclease I+ shrimp alkaline phosphatase treatment:

• In PCR tubes, add:

8.5 Ál PCR product

1 Ál Exonuclease I

1 Ál SAP (shrimp alkaline phosphatase)

• 37 deg C/20 min

• 65 deg C/20 min

VI. Sequencing

cleaned DNA

10.5 Ál

sequencing mix

8 Ál

DMSO

1 Ál

sequencing oligo –10 ÁM

0.5 Ál

• 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'


 
Prepared By:
Michael McMurray
(206) 667-6660
Last Modified: Aug 29, 2001


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