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

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1018

 

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'

 

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