PCR Problem - 300bp skip in the middle (Oct/09/2008 )
Hi someone with experience please help a noob out 
basically I am trying to insert the gene I want into a new vector, everything worked well except when my sequence came back I observe a deletion of about 300bp in the coding sequence (in the middle, beginning and end match), at first I thought maybe this is a template problem the sequence came out to be correct. So I'm just wondering if this could be a primer problem (my tm values are very high (84 and 85) due to high # of gs and cs). I only have one product that's why I suspect that it could be a primer problem becuase they can't just all "fall off" and anneal back 300bp later.
My PCR condition are as follows
0.4ug template
1ul 10uM primer 1
1ul 10uM primer 2
5ul 10x buffer
39ul h2o
2ul 10mM dntp
0.5ul pfu
94C 5min
94C 30sec
64C 30sec
72C 2min
X35
72C 10min
4C hold
I'm wondering if anyone else had encountered this "skipping" problem, any help will be greatly appreciated,
Thanks in advance,
Zip
basically I am trying to insert the gene I want into a new vector, everything worked well except when my sequence came back I observe a deletion of about 300bp in the coding sequence (in the middle, beginning and end match), at first I thought maybe this is a template problem the sequence came out to be correct. So I'm just wondering if this could be a primer problem (my tm values are very high (84 and 85) due to high # of gs and cs). I only have one product that's why I suspect that it could be a primer problem becuase they can't just all "fall off" and anneal back 300bp later.
My PCR condition are as follows
0.4ug template
1ul 10uM primer 1
1ul 10uM primer 2
5ul 10x buffer
39ul h2o
2ul 10mM dntp
0.5ul pfu
94C 5min
94C 30sec
64C 30sec
72C 2min
X35
72C 10min
4C hold
I'm wondering if anyone else had encountered this "skipping" problem, any help will be greatly appreciated,
Thanks in advance,
Zip
How come your annealing is so high? issit naturally high or issit because u added some RE site and extra few overhang?
I have encountered this kind of problems on several occasions (the joys of working with tandem array repeats in centromeres).
The problems were solved mostly by reducing the extension temperature from 72 Celsius to 68 Celsius, with a correspondingly increased extension time.
And yes, the growing DNA strand can slip... and reanneal at some other point and restart synthesis. Quite troublesome if you are working on triplet repeat expansion genes.
I would use Phusion polymerase, this polymerase (due to its dsDNA binding domain -hence the fusion in its name) allow extension of the primer at higher temperature than most other polymerase as it is less likely to fall off.
I would also recommend that you use betaine to help lower your tm.
So the template is all there but when you sequenced the PCR product it contained the deletion? Is that correct? I didn't completely understand your post. Assuming that is correct, i've never experienced that myself but it probably has something to do with the GC-richness of your gene. GC-rich sequences form strong secondary structures and "loop out", so if what Pernese says is true and the growing strand can skip sequence and re-attach, this wouldn't be too difficult if your sequence has formed a loop. Betaine dissolves secondary structures such as loops so this would probably help.
basically I am trying to insert the gene I want into a new vector, everything worked well except when my sequence came back I observe a deletion of about 300bp in the coding sequence (in the middle, beginning and end match), at first I thought maybe this is a template problem the sequence came out to be correct. So I'm just wondering if this could be a primer problem (my tm values are very high (84 and 85) due to high # of gs and cs). I only have one product that's why I suspect that it could be a primer problem becuase they can't just all "fall off" and anneal back 300bp later.
My PCR condition are as follows
0.4ug template
1ul 10uM primer 1
1ul 10uM primer 2
5ul 10x buffer
39ul h2o
2ul 10mM dntp
0.5ul pfu
94C 5min
94C 30sec
64C 30sec
72C 2min
X35
72C 10min
4C hold
I'm wondering if anyone else had encountered this "skipping" problem, any help will be greatly appreciated,
Thanks in advance,
Zip
How come your annealing is so high? issit naturally high or issit because u added some RE site and extra few overhang?
sal1 and not1 re sites are added
The problems were solved mostly by reducing the extension temperature from 72 Celsius to 68 Celsius, with a correspondingly increased extension time.
And yes, the growing DNA strand can slip... and reanneal at some other point and restart synthesis. Quite troublesome if you are working on triplet repeat expansion genes.
I would use Phusion polymerase, this polymerase (due to its dsDNA binding domain -hence the fusion in its name) allow extension of the primer at higher temperature than most other polymerase as it is less likely to fall off.
I would also recommend that you use betaine to help lower your tm.
thanks a lot I'll def try this to see if it helps solving the problem, also when you said "with a correspondingly increased extension time", could you give me a range, like if my normal extension time is 30s, maybe increase it to say like 40s?
ya that's correct. the gene is very gc rich thats one of the reason the primer annealing temp can't be dropped dramatically by tweaking w/ re.
i'll def try that method thanks.
The problems were solved mostly by reducing the extension temperature from 72 Celsius to 68 Celsius, with a correspondingly increased extension time.
And yes, the growing DNA strand can slip... and reanneal at some other point and restart synthesis. Quite troublesome if you are working on triplet repeat expansion genes.
I would use Phusion polymerase, this polymerase (due to its dsDNA binding domain -hence the fusion in its name) allow extension of the primer at higher temperature than most other polymerase as it is less likely to fall off.
I would also recommend that you use betaine to help lower your tm.
thanks a lot I'll def try this to see if it helps solving the problem, also when you said "with a correspondingly increased extension time", could you give me a range, like if my normal extension time is 30s, maybe increase it to say like 40s?
Yes, around that region. If extension time is 30 sec, I would use 45 sec. About a 50% increase.
Best of luck.
The problems were solved mostly by reducing the extension temperature from 72 Celsius to 68 Celsius, with a correspondingly increased extension time.
And yes, the growing DNA strand can slip... and reanneal at some other point and restart synthesis. Quite troublesome if you are working on triplet repeat expansion genes.
I would use Phusion polymerase, this polymerase (due to its dsDNA binding domain -hence the fusion in its name) allow extension of the primer at higher temperature than most other polymerase as it is less likely to fall off.
I would also recommend that you use betaine to help lower your tm.
thanks a lot I'll def try this to see if it helps solving the problem, also when you said "with a correspondingly increased extension time", could you give me a range, like if my normal extension time is 30s, maybe increase it to say like 40s?
Yes, around that region. If extension time is 30 sec, I would use 45 sec. About a 50% increase.
Best of luck.
kk thanks a lot man
hey guys just an update
i did it again using pernese's advice by lowering the temperature to 68 and increase the extension time to 3min. I couldn't find betaine in the lab so I used DMSO instead since it's also a polar aprotic solvent. And I think it worked because now my product increased from 750bp to around 1000bp which is close to the expected length (945bp).
Thanks everyone again for your help!