InversePCR

from 5 ml culture, resuspend in 50 µl TE

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

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

=> 5''-taagttgggtaacgccagggttttc-3''

=> 5''-ttccatgttgccactcgctttaatg-3''

=> 5''-ataactacgatacgggagggcttacc-3''

=> 5''-gattaagcattggtaactgtcagacc-3''

=> 5''-cataattctcttactgtcatgccatcc-3''

=> 5''-tcaaggatcttaccgctgttgagatcc-3''

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.

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

elute in 50 ul TE

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''