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aakashconky
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 genetics
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Posted on 09-28-11 6:15 PM     Reply [Subscribe]
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What would happen to the samples in the following PCR reactions ?

lid 85deg ; Wait; Auto
95deg 5min
95deg 30 sec
40deg 60 sec
72deg 30 sec
Goto 2 rep 40 times 
72deg 5 minutes 
Hold 4 deg

 
Posted on 09-28-11 7:33 PM     [Snapshot: 77]     Reply [Subscribe]
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 I think following happens.

lid 85deg ; Wait; Auto : preheats the PCR device to 85 deg and hold the temp at 85 

95deg 5min temp goes up to 95 and stays there for 5 min

95deg 30 sec Cycle begins. First step is to heat the sample upto 95 deg and hold it for 30 sec

40deg 60 sec Second step of the cycle. Temp is lowered to 40 deg and holds at 40 for 60 sec

72deg 30 sec THird step. Temp goes up to 72 and hols the sample for 30 sec at 72. 1 Cycle ends.

Goto 2 rep 40 times Repeats the above cycle for 40 times

72deg 5 minutes After 40 repeats brings the sample to 72 deg and holds the sample at 72 deg for 5 min.End of PCR.

Hold 4 deg Stores your sample at 4Deg. 

Hope it helps.

Sparrow
 

 
Posted on 09-28-11 9:45 PM     [Snapshot: 140]     Reply [Subscribe]
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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......

 
sYaKuuRiolAKU_nchImb
Posted on 09-28-11 10:04 PM     [Snapshot: 163]     Reply [Subscribe]
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Shiva nagar is i guess right,
40 degrees is too low for a 20 mer primer with workable proportion of GC and AT
also depends on what enzyme you are using, Taq, pfu or phusion., kappa etc  phusion requires more Tm and pfu less. In either case 40 is low

you are expected to get
1. primer binding to itself
2. more non specific product
3. no product at all

read the answers above from other users if you are looking for a basic PCR reaction mechanism


 
Posted on 09-28-11 10:29 PM     [Snapshot: 188]     Reply [Subscribe]
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Posted on 09-28-11 11:22 PM     [Snapshot: 211]     Reply [Subscribe]
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 @aakashconky timilai chine jasto lagyo...genetics class kasto bhai ra cha ta ..ha ha :P 
 
Posted on 09-28-11 11:37 PM     [Snapshot: 229]     Reply [Subscribe]
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Maile pani timilai ekchoti Mai Chine, Jaso taso chali ra cha !
 


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