Hello all,
I am in search for a PCR program for screening TDNA inserts in plants.The annealing temp of my Primer set is 61C.Could someone help me with the PCR steps i have to use for TDNA genotyping in plants?
Thanks in advance.
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bob1
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Thelymitra pulchella
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Excellent
Excellent
Posted 05 October 2012 - 01:45 PM
Your best bet will be to design the PCR program for yourself, as the exact conditions depend on the primers you use, the PCR machine (ramp times, stability), and the polymerase used.
A basic PCR program is this:
1)95 for 5 min,
2)95 for 1 min
3)Primer annealing temp for 1 min (optimise this for your primers. Note: Tm (melting temp) is not the same as Ta (annealing temp)
4)72 for 1 min)
5)repeat steps 2, 3, 4, 30x
6)72 for 5 min.
The exact extension time to use is dependent on the processivity of the polymerase, Taq will do about 1000 bp/10s.
A basic PCR program is this:
1)95 for 5 min,
2)95 for 1 min
3)Primer annealing temp for 1 min (optimise this for your primers. Note: Tm (melting temp) is not the same as Ta (annealing temp)
4)72 for 1 min)
5)repeat steps 2, 3, 4, 30x
6)72 for 5 min.
The exact extension time to use is dependent on the processivity of the polymerase, Taq will do about 1000 bp/10s.
