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Odd point mutations


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23 replies to this topic

#16 HomeBrew

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Posted 30 August 2009 - 06:40 PM

Is your -20 freezer of the frost-free variety?

#17 fishdoc

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Posted 30 August 2009 - 07:21 PM

Homebrew: The freezer is not frost-free.

Warren: Agreed on the time and money. I will be starting a couple other constructs starting this week with new polymerase, buffers, and dNTPs. The only thing that will be the same are the primers and the template. Also will try lowering the cycles. If we continue to get bad sequence, we may end up doing more to determine what the problem is. Hopefully, by changing the above things out, we'll get back on track.

#18 fishdoc

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Posted 17 September 2009 - 02:31 PM

Alright, so here's an update.

Had new polymerase, buffer, and dNTPs in hand and tried to amplify a 4.7 kb product with both Phusion and Pfu Ultra II. Neither worked, and that was odd considering it had worked fine the week before, with both polymerases. Ran some of my genomic template on a gel, and there was nothing to be seen, so I figured it had gone bad.

Did a new genomic prep, repeated the PCR with Phusion and got excellent results. Gel purified the band, inserted it, sequenced it, and everything was correct (sequenced the entire 4.7 kb piece), no point mutations.

Also, some of the other constructs I had been struggling with came out clean as well using Pfu Ultra II instead of Phusion, and using the "old" genomic DNA as template.

In the end, I'm really not sure what the hell was wrong. New genomic DNA helped get the 4.7 kb product made with Phusion (newly ordered), and it did not have any point mutations in either of the clones I sequenced. An older stock of Pfu Ultra II did better than the older stock of Phusion for the other constructs I was making, but there was still a pretty high amount of point mutations (relatively speaking). For instance, in making 2 different constructs, I sent 3 clones of each, and in both cases, 2 of 3 clones for each construct had point mutations. Luckily, there was one for each that was clean and I could continue on with.

Perhaps it was the genomic DNA that was causing the problems. It was an older prep, stored in water at 4C. Perhaps something happened to it that randomly affected a base here or there in some of the strands, resulting in mutations by the polymerase (no fault of the polymerase).

As far as I can tell, everything is back to good for now in terms of fidelity. If anything further happens, I'll be sure to update this with the details.

#19 Warren

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Posted 18 September 2009 - 08:46 AM

Alright, so here's an update.

Had new polymerase, buffer, and dNTPs in hand and tried to amplify a 4.7 kb product with both Phusion and Pfu Ultra II. Neither worked, and that was odd considering it had worked fine the week before, with both polymerases. Ran some of my genomic template on a gel, and there was nothing to be seen, so I figured it had gone bad.

Did a new genomic prep, repeated the PCR with Phusion and got excellent results. Gel purified the band, inserted it, sequenced it, and everything was correct (sequenced the entire 4.7 kb piece), no point mutations.

Also, some of the other constructs I had been struggling with came out clean as well using Pfu Ultra II instead of Phusion, and using the "old" genomic DNA as template.

In the end, I'm really not sure what the hell was wrong. New genomic DNA helped get the 4.7 kb product made with Phusion (newly ordered), and it did not have any point mutations in either of the clones I sequenced. An older stock of Pfu Ultra II did better than the older stock of Phusion for the other constructs I was making, but there was still a pretty high amount of point mutations (relatively speaking). For instance, in making 2 different constructs, I sent 3 clones of each, and in both cases, 2 of 3 clones for each construct had point mutations. Luckily, there was one for each that was clean and I could continue on with.

Perhaps it was the genomic DNA that was causing the problems. It was an older prep, stored in water at 4C. Perhaps something happened to it that randomly affected a base here or there in some of the strands, resulting in mutations by the polymerase (no fault of the polymerase).

As far as I can tell, everything is back to good for now in terms of fidelity. If anything further happens, I'll be sure to update this with the details.


Thanks for the update -- it could be the template, but I would be surprised if you could get amplification from a template that was being degraded (mutated), I might also expect the same type of mutation to be found. I am still suspicious of your old dNTPs!!! :( Warren..

#20 teresa

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Posted 03 February 2010 - 02:08 AM

Hi,

I've been having the same problem: sequencing of my plasmid constructs show several abnormal point mutations (PCR: Pfu Ultra II Fusion HS DNA Polymerase; cells: E.coli DH5alpha).

Did you manage to find out the reason for the mutations?

many thanks,

Teresa

#21 fishdoc

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Posted 03 February 2010 - 05:57 AM

Hi,

I've been having the same problem: sequencing of my plasmid constructs show several abnormal point mutations (PCR: Pfu Ultra II Fusion HS DNA Polymerase; cells: E.coli DH5alpha).

Did you manage to find out the reason for the mutations?

many thanks,

Teresa



I do not know exactly what was happening. I had the problem with both Ultra II, which had been in the freezer a couple years, and Phusion, which had been in the freezer a couple months. We ordered new aliquots of both, I purified fresh gDNA for template, and I haven't had that problem since.

#22 HomeBrew

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Posted 03 February 2010 - 07:46 AM

I'd suspect the old template -- perhaps some form of acid hydrolysis or depurination occurred due to prolonged storage in unbuffered water?

#23 fishdoc

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Posted 03 February 2010 - 08:48 AM

I'd suspect the old template -- perhaps some form of acid hydrolysis or depurination occurred due to prolonged storage in unbuffered water?



That was my thinking as well. When I made the new preps, I resuspended in tris rather than water. A colleague had just started using the enzymes for some cloning and had much less of a problem with mutations than I did. I think she had 1 point mutation in a few sequencing reactions, whereas I was getting up to 4 or 6. Have done a lot of cloning since, with both Phusion and Ultra II, and haven't had any issue with the new template.

#24 georgiadave

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Posted 04 February 2010 - 09:54 AM

I have had a similar problem. I was using gDNA as the template for PCR and looking for a 6.5kb amplicon. At first everything went as planned but over time all downstream experiments went wrong giving me zero clones (I was doing this for a couple vectors). My solution was growing fresh cells for mini-prep.




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