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Amplicon as Template in PCR for TOPO TA Cloning

PCR TOPO TA CLONING CLONING

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10 replies to this topic

#1 Epigeneticist

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Posted 01 December 2012 - 12:10 PM

I'm running low on a sample for TOPO TA cloning but have some leftover PCR product generated from this sample. I was wondering if I could use this gel purified PCR product as a template in PCR to be used for TOPO TA cloning. Will the sequence still be fully intact or will there be some issues at the 5' and 3' ends?

Thanks

#2 Trof

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Posted 01 December 2012 - 12:21 PM

The cloning is most efficient with fresh PCR template. The A overhangs degrade spontaneously over time and even those fresh ones have only some percentage of overhangs. Probably you will get lower (sometimes substantially lower) transformation efficiency (and/or negative clones unless you use ccdB vector).

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#3 Epigeneticist

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Posted 01 December 2012 - 12:29 PM

I do not want to directly use the old PCR product for TOPO TA cloning. Instead, I want to use the old PCR product as a template in a new PCR reaction then use this amplicon for TOPO TA cloning. Any suggestions?

#4 John Forsberg

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Posted 01 December 2012 - 01:31 PM

I don't see why doing your PCRs off of a previous product will not work perfectly well. I've done PCRs off of gel-extracted PCR product bands before (to avoid non-specific priming of my plasmid) in conventional restriction cloning and it worked beautifully. In your case, your polymerase shouldn't have any problem adding the A overhangs. You don't have to worry about your previous product being degraded either, as your PCR amplification will select for full-length templates.

I just used 1 µl of my gel extraction (which was probably massive overkill), but you could probably use even less than that to make sure you don't get partial ligations with your "template" that may only have an A overhang on one end.

#5 Trof

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Posted 01 December 2012 - 02:26 PM

CJB: I see. You can with only one, but particulary important drawback. PCR with Taq polymerases causes mutations, the same polymerase that creates A overhangs needed for TA sequencing. So unles you use a mix of polymerases or add overhangs after PCR you increase the risk of missense in your clone, which can be actually pretty high (the Taq polymerase I use for common sequencing and never give me a false read when used for cloning, only one in four inserts I completely sequenced after TA cloning had no mutations). So if you do PCR on a PCR product, the rate of mutations may be even higher.

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#6 phage434

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Posted 01 December 2012 - 02:42 PM

Short PCR fragments, sometimes barely visible on a gel, can dominate the desired template in a second round of PCR with the same primers. I usually expect problems when I do this, even after gel purification. Remember that a barely visible band at 100 bp has many many more copies than a much brighter band at 4000 bp.

#7 Epigeneticist

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Posted 01 December 2012 - 02:52 PM

I guess the risk of single point mutations is not too big of a problem for me. The TOPO TA cloning is for bisulfite sequencing. As long as the mutation(s) are other than the C​pGs I'm trying to interrogate or if they do hit the CpG they are a C/T =>G or or C/T=>A mutation.

#8 Epigeneticist

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Posted 01 December 2012 - 02:59 PM

phage434

"I usually expect problems when I do this, even after gel purification.Remember that a barely visible band at 100 bp has many many more copies than a much brighter band at 4000 bp." Why would there still be a problem after gel purification in your example? Gel purification would exclude the 100bp band from the 4kb. Maybe I'm not understanding. I understand the risk of mutations now but not sure about shorter length products causing a problem after gel purification prior to the next round of PCR.

Mine
1. Product size = 505bp
2. Product is gel purified

I would only be amplifying the 505bp on the second PCR reaction.

#9 phage434

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Posted 01 December 2012 - 03:04 PM

Gel purification is far from perfect, and leaves short fragments. The short fragments amplify in PCR better than longer ones, and are perfect matches for both primers, just like your desired amplicon. Sometimes it works.

#10 Epigeneticist

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Posted 01 December 2012 - 03:25 PM

One step further just for clarification - when you refer to short do you mean:

For example in my case with a 505bp product and gel purification,

1- Gel purification of my desired band would give me a 505bp product along with some other products such as 500bp, 495bp etc

or

2 - gel purification of my desired product would give me a 505bp product along with some very short products such as 100bp which can sometimes be amplified very efficiently.

I understand how I could gel purify a product similar in size to my 505bp product but I'm not sure how I would end up purifying something like a 100bp band with a 505bp band. Would some residual 100bp product somehow co-migrate with the 505bp band or something? Even though its only a small amount, it efficiency of amplication is so high that it causes problems? Just looking for an explanation to expand my knowledge.

Thanks,

#11 Trof

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Posted 01 December 2012 - 03:34 PM

I don't think you can get shorter fragments (like 100 bp) if you cut 505 bp from gel. I rePCRd gel-purified products several times, usually for sequencing, and it was fine in all cases. Cloning is more sensitive to residual short fragments than sequencing, that's true, but you can easily select the right clones by colony PCR even before you grow them in minipreps.

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon






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