Help! illumina sequencing sample size shifted in Bioanalyzer?

RUmustang · 04-16-2010, 09:42 PM

I am currently working on illumina sequencing sample preparation. For final PCR product, I check the same samples using invitrogen E-gel and bioanalyzer, but the DNA size from Bioanalyzer is always around 100bp larger than that on E-gel. I cut the 250bp band out of gel and qiagen column purified it, but I got a 350-400bp peak in Bioanalyzer. I am wondering if anybody here had the same experience and if this sample could still be sequenced. Thanks a lot.

LMcSeq · 04-17-2010, 10:12 AM

Try cleaning the bayonette cartridge in the bioanalyzer and rerunning first. We sometimes get shifts in samples and it's just because it needs a good cleaning. Soak it in molecular grade water overnight and then see what the run looks like.

RUmustang · 04-17-2010, 11:09 PM

thanks for the reply. Actually I run the E-gel DNA ladder and a regular PCR product (not an illumina sample) in the bioanalyzer. Both sizes are correct. I guess maybe my illumina samples contain some sort of complicated secondary structures. BTW, I forget to mention that the primers I used are not from illumina. I don't know if the illumina primers are modified so that they will give me the clean PCR product.

glucoses14 · 04-22-2010, 01:48 PM

Hi,
I am glad I am not the only one to get this. I also use none-illumina primer. At first, my libraries are fine and recently, the size shifted about 100bp up and I also suspect this is an artificial library. May I ask if what PCR reagent you are using? 2X phusion master mix or following the illumina protocol by adding enzyme, buffer, dNTP separately?

thanks d for the discussion.

RUmustang · 04-22-2010, 03:35 PM

I used phusion polymerase and added those reagents separately. Additionally, I don't think it's phusion's issue since I tried pfu as well and got similar products. I guess we got complicated secondary structures in our PCR product (not just linear double stranded DNA) which cause either gel or bioanalyzer size shift.

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04-16-2010, 09:42 PM	#1
RUmustang Junior Member Location: walnut creek, CA Join Date: Apr 2010 Posts: 4	Help! illumina sequencing sample size shifted in Bioanalyzer? I am currently working on illumina sequencing sample preparation. For final PCR product, I check the same samples using invitrogen E-gel and bioanalyzer, but the DNA size from Bioanalyzer is always around 100bp larger than that on E-gel. I cut the 250bp band out of gel and qiagen column purified it, but I got a 350-400bp peak in Bioanalyzer. I am wondering if anybody here had the same experience and if this sample could still be sequenced. Thanks a lot.

04-22-2010, 01:48 PM	#4
glucoses14 Junior Member Location: CA Join Date: Nov 2009 Posts: 1	I got the same problem recently Hi, I am glad I am not the only one to get this. I also use none-illumina primer. At first, my libraries are fine and recently, the size shifted about 100bp up and I also suspect this is an artificial library. May I ask if what PCR reagent you are using? 2X phusion master mix or following the illumina protocol by adding enzyme, buffer, dNTP separately? thanks d for the discussion.

04-17-2010, 10:12 AM	#2
LMcSeq Member Location: Maryland--we have a HiSeq too! Join Date: Feb 2009 Posts: 33	Try cleaning the bayonette cartridge in the bioanalyzer and rerunning first. We sometimes get shifts in samples and it's just because it needs a good cleaning. Soak it in molecular grade water overnight and then see what the run looks like.

04-17-2010, 11:09 PM	#3
RUmustang Junior Member Location: walnut creek, CA Join Date: Apr 2010 Posts: 4	thanks for the reply. Actually I run the E-gel DNA ladder and a regular PCR product (not an illumina sample) in the bioanalyzer. Both sizes are correct. I guess maybe my illumina samples contain some sort of complicated secondary structures. BTW, I forget to mention that the primers I used are not from illumina. I don't know if the illumina primers are modified so that they will give me the clean PCR product.

04-22-2010, 03:35 PM	#5
RUmustang Junior Member Location: walnut creek, CA Join Date: Apr 2010 Posts: 4	I used phusion polymerase and added those reagents separately. Additionally, I don't think it's phusion's issue since I tried pfu as well and got similar products. I guess we got complicated secondary structures in our PCR product (not just linear double stranded DNA) which cause either gel or bioanalyzer size shift.