Griffitts:Arbitrary PCR

Introduction

You need nested outward-pointing transposon-specific primers (TSP) within 150 bp of the transposon end. These should both be standard primers with T_m 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

Recipe

• 21.35 μL dH₂O
• 2.5 μL Taq buffer
• 0.6 μL 10mM dNTPs
• 0.15 μL 100uM TSP1 (for mini-Tn5:110 use oJG133)
• 0.15 μL 100uM ARB1A or ARB1B
• 0.25 μL Taq (NEB works nicely)
• 1 μL template (boiled cells)

Note: For amplifying rhizobium sequences, boil a dense suspension of cells in 35 μL PCR lysis buffer for ~2 min and vortex; for amplifying E. coli, adding cells directly to the reaction is sufficient

Cycling

1. 94°C for 3:00
2. 94°C for 0:20
3. 33°C for 0:20
4. 70°C for 1:00
5. GOTO 2, 6 times
6. 94°C for 0:20
7. 43°C for 0:20
8. 70°C for 1:00
9. GOTO 6, 28 times
10. 70°C for 3:00
11. 4°C forever

Note: For more info, see the standard PCR procedure

Round Two

Recipe

• 21.35 μL dH₂O
• 2.5 μL Taq buffer
• 0.6 μL 10mM dNTPs
• 0.15 μL 100uM TSP2 (for mini-Tn5:110 use oJG134)
• 0.15 μL 100uM ARB2
• 0.25 μL Taq
• 0.8 μL first-round rxn.

Cycling

1. 94°C for 3:00
2. 94°C for 0:20
3. 52°C for 0:20
4. 70°C for 1:30
5. GOTO 2, 30 times
6. 70°C for 3:00
7. 4°C forever

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