SEQanswers

Go Back   SEQanswers > Sequencing Technologies/Companies > Illumina/Solexa



Similar Threads
Thread Thread Starter Forum Replies Last Post
PCR primers+adaptors gio5 Sample Prep / Library Generation 2 01-05-2012 09:32 AM
How to eliminate PE dimers from final product? OnUrMark RNA Sequencing 11 06-02-2011 04:38 AM
PCR duplicates increase when excess of beads tdm SOLiD 10 03-31-2011 08:48 AM
PCR product normalization Palecomic Sample Prep / Library Generation 0 01-20-2011 01:50 AM
Request for sequencing of PCR product Damann Core Facilities 0 11-05-2009 07:34 AM

Reply
 
Thread Tools
Old 10-02-2008, 03:28 PM   #1
PaulineF
Member
 
Location: Canada

Join Date: Jul 2008
Posts: 15
Default Excess PCR primers in final product

Hi,

Has anyone ever had primers that were too concentrated for the paired-end sample prep? We are getting excess primers left over after the PCR, which is after the gel purification. But the excess primers were not there before the PCR. Moreover, our first batch of PE sample preps were fine (i.e. no excess primers).

PF
PaulineF is offline   Reply With Quote
Old 10-02-2008, 07:20 PM   #2
ECO
--Site Admin--
 
Location: SF Bay Area, CA, USA

Join Date: Oct 2007
Posts: 1,358
Default

I don't understand your question. Sounds like you got primer-dimer or non-specific products, rather than primers that were too concentrated.
ECO is offline   Reply With Quote
Old 10-03-2008, 10:46 AM   #3
PaulineF
Member
 
Location: Canada

Join Date: Jul 2008
Posts: 15
Default

The whole story was the new PE protocol says to run 10 cycles of PCR and when I first did that, all I got was an amplification of primers because the cycles were not long enough to amplify the actual sample. Then I increased the cycle number to 16 and saw my sample smear between 200-350 bp but in addition, there was a band around 77bp that is consistent across all samples corresponding to the primer peak. The point is we have run similar samples through another PE kit before and never got the extra primer peak. I ran the remainder of that old primer with the new primer on the Bioanalyzer and found the new primer to be aobut 5x more concentrated, but the readings between the various tubes of new primer varied a little.

My question is if anyone has had experience with primers that may seem too concentrated, resulting in excess amounts in the PCR product. Do you know what the stock concentrations of PE primers are?
PaulineF is offline   Reply With Quote
Old 10-08-2008, 05:40 AM   #4
suludana
Member
 
Location: Spain

Join Date: May 2008
Posts: 61
Default Primers amplification

Hi,
the same thing happened to me. And then, when I cleaned the PCR reaction (Qiagen 28104) I lose the entire smear of 100-200 bp, leaving only some of the band corresponding to the primers. What should I do?
suludana is offline   Reply With Quote
Old 10-08-2008, 06:07 AM   #5
PaulineF
Member
 
Location: Canada

Join Date: Jul 2008
Posts: 15
Default

Hi Suludana,

May I ask what are the lot numbers of your primers in question? When is the expiration date of your kit? Just curious. I had my lots checked by Illumina's R&D and they claimed those lots were fine. I tried decreasing the primer concentration going into my 18-cycle PCR reaction but only some samples had product, and for those, there was no more primer peak on the Bioanalyzer.

Pauline
PaulineF is offline   Reply With Quote
Old 10-08-2008, 06:24 AM   #6
suludana
Member
 
Location: Spain

Join Date: May 2008
Posts: 61
Default

Well, actually I was making a handmade sample preparation for indexing. I was not using an Illumina kit, which does not yet exist for indexing. But Illumina gave me the concentration of primers (25 uM) and I was working with it.

I find it very strange that the primers are positioned at about 100 bp and the higher band get away when I clean the PCR.
suludana is offline   Reply With Quote
Old 10-08-2008, 11:15 AM   #7
PaulineF
Member
 
Location: Canada

Join Date: Jul 2008
Posts: 15
Default

I was getting what is most likely primer dimers at around 77bp, that's probably what you have too. But I do standardly put my PCR product through the Qiagen kit to purify it (as per the Illumina protocol) and I don't lose my sample that way.
PaulineF is offline   Reply With Quote
Old 10-08-2008, 12:07 PM   #8
suludana
Member
 
Location: Spain

Join Date: May 2008
Posts: 61
Default

Hi,
why do you talk about primer dimers of 77 bp? How can you explain this size? One of the primers have 58 bp and the other 34 bp. And they are not complementary.
suludana is offline   Reply With Quote
Old 10-08-2008, 01:02 PM   #9
PaulineF
Member
 
Location: Canada

Join Date: Jul 2008
Posts: 15
Default

Hi,

I've been working with the Illumina kits and I am not indexing. I do not have the exact size of the primers but primer dimer was also suggested by Illumina to be what my smaller bands may be as they claim the primers are over 30nt long. I am sure though that primers for multiplexing are different from single sample PE primers. So I am not sure if you are having the same problem that I am.
PaulineF is offline   Reply With Quote
Old 10-08-2008, 01:50 PM   #10
suludana
Member
 
Location: Spain

Join Date: May 2008
Posts: 61
Default

It is true that I am doing indexed, but I'm using primers of the standard protocol (58 and 34 bp). The difference is only in 1 nucleotide (I do not have tags on my PCR primers) ... so the size is almost the same ... I see my primers in a position of 100 bp and I don't understand it ...
suludana is offline   Reply With Quote
Old 10-15-2008, 09:32 AM   #11
cjohns
Junior Member
 
Location: Seattle

Join Date: Sep 2008
Posts: 7
Default

I have done several chip-seq with illumina kits and on my last set I also got a product that is about 80bp long that I have not seen before. It is in my blank gel that I amplified too, so it has to be from the amplification primers. My primers from different kits seem to have similar concentrations according to the spec I used. It is very odd that all of a sudden I am getting primer dimers now. Has anyone figured out how to fix this problem?
cjohns is offline   Reply With Quote
Old 10-15-2008, 01:50 PM   #12
PaulineF
Member
 
Location: Canada

Join Date: Jul 2008
Posts: 15
Default

I heard that somehow in the gel purification step, if you use the Qiagen kit as Illumina suggested, instead of heating your gel+buffer mix at 50C, vortex at high speed for 5 mins at RT and that somehow helps prevent concatemers. I have yet to try it out, but I heard it works.

But yes, I suddenly started to get these small peaks too, samples are similar enough to what I've run before and we're still supposedly buying the same kits. Weird...
PaulineF is offline   Reply With Quote
Old 03-03-2009, 12:31 PM   #13
mjg54
Junior Member
 
Location: ithaca ny

Join Date: Mar 2009
Posts: 2
Default does the band get sequenced?

I completed my first sample prep and I see a band just below 100bp, I think it is an amplicon product from the primers priming one another. I was wondering if this will be sequenced, because it is so short it may not undergo bridge amplification.
mjg54 is offline   Reply With Quote
Old 03-03-2009, 12:47 PM   #14
cjohns
Junior Member
 
Location: Seattle

Join Date: Sep 2008
Posts: 7
Default

No, they do not get sequenced. Our only concern is we use PicoGreen to quantitate and it would detect these amplicon products and we may not have reliable quantitation of our insert DNA.
cjohns is offline   Reply With Quote
Old 03-03-2009, 12:55 PM   #15
mjg54
Junior Member
 
Location: ithaca ny

Join Date: Mar 2009
Posts: 2
Default what are they

so, what is the 100bp band?
I can estimate from a gel what percentage of my sample is the "sequencable" smear and what is the 100 bp band, then when I quantify I can normalize.

thanks for the extremely quick response
mjg54 is offline   Reply With Quote
Old 03-06-2009, 10:11 AM   #16
BioHak
Member
 
Location: PharmLand -- Philadelphia

Join Date: Aug 2008
Posts: 13
Default

If you believe it is an amp by-product, why not sequence a sample of it on a capillary instrument and verify?

If your goal is to remove the smaller fragment, we cleanup our samples with the Aggencourt AmPure product? We see significant dropoff of any products under ~90bps and nearly 100% recovery.
BioHak is offline   Reply With Quote
Old 06-18-2009, 04:55 PM   #17
captainentropy
Member
 
Location: San Francisco Bay Area

Join Date: Mar 2009
Posts: 89
Default

Unless there is something going on that I haven't figured out yet I think the peaks you are describing for the PE amplification are amplified primers as I am seeing them too. However, the size of the peaks are larger, around 125 bp, though there is a tiny peak around 66 bp as well. In previous libraries I've seen a very strong 150-160bp peak that I know was amplified adapters that resulted from me cutting too far down on the gel after the ligation step. I've already sent these current samples for sequencing, I'll know for sure in a week or two whether they are adapters or primers.

In the attached images are my last two libraries. One is a library made from 100ng of input DNA and the other I used 1000 ng. Other than that and the day I made them on they are identical in construction (also, I use a 1:30 dilution of adapter instead of 1:10). Another person in my lab is using the same paired-end protocol and he has seen the same pattern. It was also present in a no-template control reaction, so we're confident it's just amplified primers. He has been optimizing the PCR step and has found that raising the annealing temp didn't prevent the peak from appearing and neither did adding DMSO to the reaction. The only thing that worked for him was to optimize the primer concentration. The concentration of the primers Illumina provides (or tells you to use if you ask them) is 25 uM. Thus in the PCR the [final] = 1 uM. For standard PCR I've always used [final] = 0.4 uM, and that is for 25 or more cycles! Although I am using the PE adapters/primers I've been using 18 cycles of PCR as written for the single-end adapter protocol. I'm not sure if I used 12 cycles as written for the PE protocol that it would make much difference as the products amplified in the first few cycles are most important for the final outcome. I can always just run the PCR on a gel and purify the library from the primers but the problem is at the PCR step. I think the primer concentration Illumina is suggesting is too high. Any opinions on that?
Attached Images
File Type: jpg library-100ng (Medium).jpg (39.8 KB, 213 views)
File Type: jpg library-1000ng (Medium).jpg (39.4 KB, 174 views)

Last edited by captainentropy; 06-22-2009 at 12:15 PM. Reason: forgot to add an important point
captainentropy is offline   Reply With Quote
Old 06-20-2009, 05:41 AM   #18
csoong
Member
 
Location: Connecticut

Join Date: Jun 2009
Posts: 74
Default

The pre-PCR gel extracted product should be characterized in terms of OD ratio and concentration and this would act as a validation step prior to PCR. This pre-PCR quality may greatly influence the required amount of starting materials and number of thermal cycles required for PCR-ing. If we could share info about pre-PCR characterization, post PCR characterization, insert size, and PCR settings on successful sequence runs, this would be very helpful to the community.
csoong is offline   Reply With Quote
Old 06-22-2009, 02:56 PM   #19
captainentropy
Member
 
Location: San Francisco Bay Area

Join Date: Mar 2009
Posts: 89
Default

agreed csoong. However, if you are using ChiPed DNA you might only have a few nanograms to work with. That is hard too quantitate much less get a 260/280 ratio.

In my lab we're pretty sure the peaks we are/were seeing come from the PCR primers. Once I get my sequence back I should know for sure though.
captainentropy is offline   Reply With Quote
Old 07-27-2010, 04:23 AM   #20
JUdw
Junior Member
 
Location: The Netherlands

Join Date: Dec 2009
Posts: 9
Default

Quote:
Originally Posted by captainentropy View Post
....The concentration of the primers Illumina provides (or tells you to use if you ask them) is 25 uM. Thus in the PCR the [final] = 1 uM. For standard PCR I've always used [final] = 0.4 uM, and that is for 25 or more cycles!
1ul [25uM] in 50ul PCR reaction, [final] = 0.5uM
JUdw is offline   Reply With Quote
Reply

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off




All times are GMT -8. The time now is 03:20 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2020, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO