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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 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 NH₄Ac + 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

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'

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

0.5 µl

• 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