Griffitts:Arbitrary PCR: Difference between revisions
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==Round One== | ==Round One== | ||
=== | ===Round One recipe=== | ||
{| border="1" cellpadding="5" cellspacing="0" | |||
|- | |||
! width="120" style="background:#efefef;" | Ingredient | |||
! width="120" style="background:#efefef;" | Per reaction | |||
|- | |||
| dH<sub>2</sub>O | |||
| 21.35 μL | |||
|- | |||
| [[Griffitts:Stock solutions#10X Taq buffer|Taq buffer]] | |||
| 2.5 μL | |||
|- | |||
| [[Griffitts:Stock solutions#10 mM dNTPs|10mM dNTPs]] | |||
| 0.6 μL | |||
|- | |||
| 100uM TSP1 (for mini-''Tn5'':110 use oJG133) | |||
| 0.15 μL | |||
|- | |||
| 100uM [[Griffitts:Arbitrary PCR#ARB1A|ARB1A]] or [[Griffitts:Arbitrary PCR#ARB1B|ARB1B]] | |||
| 0.15 μL | |||
|- | |||
| Taq (NEB works nicely) | |||
| 0.25 μL | |||
|- | |||
| DNA template (boiled cells) | |||
| 1 μL | |||
|} | |||
<br> | |||
Note: For amplifying rhizobium sequences, boil a dense suspension of cells in 35 μL [[Griffitts:Common buffers#colony lysis solution (for PCR) (100 mL)|PCR lysis buffer]] for ~2 min and vortex; for amplifying ''E. coli'', adding cells directly to the reaction is sufficient | Note: For amplifying rhizobium sequences, boil a dense suspension of cells in 35 μL [[Griffitts:Common buffers#colony lysis solution (for PCR) (100 mL)|PCR lysis buffer]] for ~2 min and vortex; for amplifying ''E. coli'', adding cells directly to the reaction is sufficient | ||
Revision as of 12:09, 5 August 2009
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
| Ingredient | Per reaction |
|---|---|
| dH2O | 21.35 μL |
| Taq buffer | 2.5 μL |
| 10mM dNTPs | 0.6 μL |
| 100uM TSP1 (for mini-Tn5:110 use oJG133) | 0.15 μL |
| 100uM ARB1A or ARB1B | 0.15 μL |
| Taq (NEB works nicely) | 0.25 μL |
| DNA template (boiled cells) | 1 μL |
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
- 94°C for 3:00
- 94°C for 0:20
- 33°C for 0:20
- 70°C for 1:00
- GOTO 2, 6 times
- 94°C for 0:20
- 43°C for 0:20
- 70°C for 1:00
- GOTO 6, 28 times
- 70°C for 3:00
- 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
- 94°C for 3:00
- 94°C for 0:20
- 52°C for 0:20
- 70°C for 1:30
- GOTO 2, 30 times
- 70°C for 3:00
- 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
