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Problems with sequencing


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#1 Nebulus

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Posted 03 June 2010 - 10:35 PM

Hi, I am actually having a few problems trying to sequence a gene.

First of all I have tried universal primers for my gene, and genus specific primers for my gene and I have gotten 2 bands in both cases. My desired band is 1100 bp but I always get another band that is 1000-1100 bp as well as the one of desired size. I have speculated that my gene has been duplicated and the duplicated gene has a 20-50 bp deletion. I am very confident my primers are specific.

The problem arises when I try to separate the bands for gel purification. I can't seem to get the 2 bands far enough apart without running them forever and loosing a lot of band intensity.

Any recommendations for separating 2 bands of ~1kb in size and 50 bp difference?


Secondly I am having trouble with my gel purification. My PCR products have a concentration of ~150ng yet I get very little yield (0.7-5ng/ul) after gel extraction. My 260/280 nanodrop readings are also very screwy. I use a qiagen gel purification kit. I have tried purifying my products multiple times and all show about the same thing. I've spent almost a month on this and I am starting to loose my sanity.


Thanks

#2 Rsm

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Posted 04 June 2010 - 12:09 AM

Hi,


IMHO, there's no easy way to separate 1000bp and 1050bp PCR products on gel. Do you need to separate them by gel? I would suggest you to clone your PCR product directly into a TA vector (or alternatively use RE site containing primer) and sequence the bacterial colonies. You can run a PCR first, to sequence some with 1kb and some with 1.05kb insert.

Then you'll also don't need to bother with yield after gel purification. In my experience, you'll lose around 25-50% of your input. Please remember that your primer and nucleotides from your PCR reaction are still contained in your PCR products, if you measure them before purification. They will increase your reading a lot.


Cheers,

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#3 phage434

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Posted 04 June 2010 - 01:44 PM

first thing I would do is to purify the pcr product with a column and then sequence with each of the primers. You will quickly know if there are two sequences, and perhaps where they differ. If there are, I would clone them into vectors, and do colony pcr to identify the two different clones, then sequence the clones.

#4 Nebulus

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Posted 05 June 2010 - 07:53 PM

first thing I would do is to purify the pcr product with a column and then sequence with each of the primers. You will quickly know if there are two sequences, and perhaps where they differ. If there are, I would clone them into vectors, and do colony pcr to identify the two different clones, then sequence the clones.



Could you elaborate a little? This is my first sequencing attempt. How will I know if there are 2 sequences? Will there be a messed up region where the deletion occured in the gene duplication in the sequence readout?

Thanks

#5 Trof

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Posted 07 June 2010 - 05:40 AM

Could you elaborate a little? This is my first sequencing attempt. How will I know if there are 2 sequences? Will there be a messed up region where the deletion occured in the gene duplication in the sequence readout?

Yes, in the part where sequences are the same you will see simple, readable peaks. From the point of deletion/duplication there will be overlay of two sequences, that are not readable. If you sequence from both sides you can determine where the deletion/duplication starts and ends (of course only if it's in the middle of the sequence, not on the ends), and you can even substract the sequence from the known one.

If you get completely unreadable sequence, then there is a possibility, that you have unspecific amplification.

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#6 Sagesigh

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Posted 10 June 2010 - 07:12 PM

We could try to resolve and extract it for you in our lab. We are developing a range of size selection cassettes for our automated gel purification and electro-elution instrument, Pippin Prep, that should be able to parse the two sizes and hand back the size you want. We are looking for numerous samples to test with new cassettes in development. If you have interest, then please drop me line at gary@sagescience.com.




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