Griffitts:Arbitrary PCR

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Contents

Introduction

You need nested outward-pointing transposon-specific primers (TSP) within 150 bp of the transposon end. These should both be standard primers with Tm of 60–65°C. The primer more distal from the transposon end (TSP1) is to be used in the first-round PCR, and the primer more proximal to the transposon end (TSP2) is to be used in the second round PCR. You will also need a degenerate arbitrary primer designed to hybridize promiscuously at low annealing temperatures. I recommend the following two primers: ARB1A, ARB1B, or ARB1C. These would not be used simultaneously, but rather in parallel experiments to maximize the odds of getting good product. These primers are used in the first-round PCR. Finally, you need the ARB2 primer, designed to hybridize to the products of ARB1A, ARB1B, or ARB1C in the second round PCR.

Round One

Round One recipe

Per reaction Ingredient
21.35 μL dH2O
2.5 μL Taq buffer
0.6 μL 10mM dNTPs
0.25 μL Taq polymerase
0.15 μL 100μM TSP1*
0.15 μL 100μM ARB1A or ARB1B
1 μL DNA template (boiled cells)**


*For mini-Tn5:110 (NmR) use oJG133.
For mini-Tn5:267 or mini-Tn5:285 (both TcR) use oJG697.
For mini-Tn5:520 (GmR) use oJG1254.

**For amplifying rhizobium sequences, boil a dense suspension of cells in 50 μL PCR lysis buffer for ~2 min and vortex; for amplifying E. coli, adding cells directly to the reaction is sufficient.

Round One cycling

Step Temp Duration
1 94°C 3:00
2 94°C 0:20
3 33°C 0:20
4 70°C 1:00
5 GOTO 2 6 times
6 94°C 0:20
7 43°C 0:20
8 70°C 1:00
9 GOTO 6 28 times
10 70°C 3:00
11 4°C forever
12 END


Note: For more info, see the standard PCR procedure

Round Two

Round Two recipe

Per reaction Ingredient
21.35 μL dH2O
2.5 μL Taq buffer
0.6 μL 10mM dNTPs
0.25 μL Taq polymerase
0.15 μL 100μM TSP2*
0.15 μL 100μM ARB2
0.8 μL first-round rxn


*For mini-Tn5:110 (NmR) use oJG134.
For mini-Tn5:267 or mini-Tn5:285 (both TcR) use oJG698.
For mini-Tn5:520 (GmR) use oJG1255.

Round Two cycling

Step Temp Duration
1 94°C 3:00
2 94°C 0:20
3 52°C 0:20
4 70°C 1:30
5 GOTO 2 30 times
6 70°C 3:00
7 4°C forever
8 END


Note: For more info, see the standard PCR procedure

Conclusion

Second-round reactions are then cleaned up by Qiagen. Run a small amount on a gel to analyze products, and use remaining product for sequencing, using the TSP2 primer. Even if the reaction yields multiple bands, you should get unambiguous sequence back. If you are using native Tn5, in which case TSP1 and TSP2 will hybridize on both sides, then you would need to clone before sequencing.

Primers

ARB1A

GCCACGCGTCGACTAGTACNNNNNNNNNNACGCC

ARB1B

GCCACGCGTCGACTAGTACNNNNNNNNNNTGCGG

ARB1C

GCCACGCGTCGACTAGTACNNNNNNNNNNTCCGG

ARB2

GCCACGCGTCGACTAGTAC

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