Posted by: shivanagar September 28, 2011
Login in to Rate this Post:
0
?
I guess question is what happens to samples biochemically..rather than just heat and cool down.
So, Before I go on .I assume you have DNA template......Forward and reverse oligos, polymerase and dNTPs at right amount (oh ya, buffer, mg++ too),
then,
lid 85deg ; Wait; Auto--> Pre heat.and lid is always at 85, while the samples are at varied temp
95deg 5min--> denature the DNA
95deg 30 sec-->denature the DNA
40deg 60 sec-->>attach the forward and reverse oligo's , It may actually vary according to melting temp of oligo, isn't 40 too low...may get some unwanted items tooo
72deg 30 sec-->extention , work of polymerase to extend oligo by adding DNTPs
Goto 2 rep 40 times -> repeat 40 times, so, 2**40 fragments.
72deg 5 minutes -->> final extention and stabalise
Hold 4 deg--> hold for temporary storage, in case you don't come back in time to store the plate in refrigerator after you go for some coffee or beer......
So, Before I go on .I assume you have DNA template......Forward and reverse oligos, polymerase and dNTPs at right amount (oh ya, buffer, mg++ too),
then,
lid 85deg ; Wait; Auto--> Pre heat.and lid is always at 85, while the samples are at varied temp
95deg 5min--> denature the DNA
95deg 30 sec-->denature the DNA
40deg 60 sec-->>attach the forward and reverse oligo's , It may actually vary according to melting temp of oligo, isn't 40 too low...may get some unwanted items tooo
72deg 30 sec-->extention , work of polymerase to extend oligo by adding DNTPs
Goto 2 rep 40 times -> repeat 40 times, so, 2**40 fragments.
72deg 5 minutes -->> final extention and stabalise
Hold 4 deg--> hold for temporary storage, in case you don't come back in time to store the plate in refrigerator after you go for some coffee or beer......