Inverse PCR
I.
• 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
0.5µl
• 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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