How to amplify 4.1Kb gene from genomic DNA? - (Nov/13/2007 )
the intent of overlapping segments is to bind complementary ends to each other and extend along the resulting construct (in both directions) finally reproducing the gene with the desired sequence.
I wasn't saying insert new restriction sites, clearly that will alter the sequence, I said "design the overlap regions so that they contain unique RE sites". In other words, find RE sites that are unique to your insert and design the regions of overlap on top of them. This is not absolutely necessary but just provides an alternate way to achieve the desired result. The more strategies you have in place to achieve the desired result, the more likely you will get the desired result because if one strategy fails you have the other/s to fall back on.
best is ofcourse, if a polymerase can amplify the whole 4.1kb.
otherwise, it seems like one will have to do it in pieces and put them together in an overlapping pcr approach.
There are always plenty of unique restriction sites in the insert, just think about the data you get when you put your sequence into restrictionmapper and you see all the RE sites in your insert, there are always plenty of single cut sites. This approach is not as tricky as it seems and it also gives you the option of splice overlapping some pieces together and ligating others depending on how successful the splice overlaps are.
It's not so easy. Because of several reasons.
- some restriction enzymes are very rarely used and therefore difficult to find in routine laboratories (a practical problem).
- if a 4.5 kb gene is amplified as 3 pieces A, B, C each 1.5kb approximately, then it will become very difficult to distinguish A+B dimer from A+A dimer on a gel.
So, I agree that in principle it is a workable technique, but it can be equally difficult or easy depending on several factors, as are several of our other experiments.
Nevertheless, thanks for your time and comments.
Rami
the intent of overlapping segments is to bind complementary ends to each other and extend along the resulting construct (in both directions) finally reproducing the gene with the desired sequence.
I wasn't saying insert new restriction sites, clearly that will alter the sequence, I said "design the overlap regions so that they contain unique RE sites". In other words, find RE sites that are unique to your insert and design the regions of overlap on top of them. This is not absolutely necessary but just provides an alternate way to achieve the desired result. The more strategies you have in place to achieve the desired result, the more likely you will get the desired result because if one strategy fails you have the other/s to fall back on.
best is ofcourse, if a polymerase can amplify the whole 4.1kb.
otherwise, it seems like one will have to do it in pieces and put them together in an overlapping pcr approach.
There are always plenty of unique restriction sites in the insert, just think about the data you get when you put your sequence into restrictionmapper and you see all the RE sites in your insert, there are always plenty of single cut sites. This approach is not as tricky as it seems and it also gives you the option of splice overlapping some pieces together and ligating others depending on how successful the splice overlaps are.
Hope it helps 
- some restriction enzymes are very rarely used and therefore difficult to find in routine laboratories (a practical problem).
- if a 4.5 kb gene is amplified as 3 pieces A, B, C each 1.5kb approximately, then it will become very difficult to distinguish A+B dimer from A+A dimer on a gel.
So, I agree that in principle it is a workable technique, but it can be equally difficult or easy depending on several factors, as are several of our other experiments.
Nevertheless, thanks for your time and comments.
Rami
Yeah, restrictionmapper does spit out some real doozies, I'll give you that. Nevertheless, there are usually others to choose from. As for the 3 pieces, they don't need to be 1.5 kb each, they probably wouldn't be any way, but I'd to a 3-way ligation rather than running out the fragments on a gel, the yield is too low. You can do sequential ligations as well if you're lucky enough to have the sites you require in the right places. No worries for the comments, I like helping out - and learning - where I can. This is a great forum for that.
Good luck,
Rob
Hi
I use Ampli TAq from stratagene for genomic DNA , when Pfu polymerase does not work..
Also instead of rePCRing from the three PCRs I wud clone them individually in diff vectors
and then put them one after the other..(I guess u want to clone the gene) by using silent
restriction sites engineered in primers. This is bcoz long PCRs are not error free
gud luck
iProof polymerase from BioRad worked great! It amplified the 4.1Kb gene in the first attempt.
I tried 5 different conditions.
Reaction with added magnesium and with the GC buffer meant for high GC containing templates worked great.
Thanks folks for all your comments.
Just thought that I'll share my result with you all.
best wishes