
#1
Posted 03 September 2004 - 11:47 AM
I was wondering if anyone has answers as to why my PCR product is one big streak from well to end. Somtimes a band shows up where expected but in the background (or foreground) is the large, sometimes bright streak. We've been having this problem for a while. Sometimes it's not there but nothing different was done with extraction or PCR parameters. We use optimal thermocycling conditions for our primers, so we're pretty sure it's not in the PCR reaction.
We've also encountered bright bands in wells followed by those streaks of some samples. Any suggestions would be greatly appreciated!
Thank you in advance.
kudna
#2
Posted 08 September 2004 - 11:42 PM
I have had this problem before in the lab. But we realised that it was because People in the lab were too lazy to change the TAE buffer that was used to run the gel.
Hope this helps!
Kiwi
#3
Posted 13 September 2004 - 02:03 PM
We change our TAE every 5 runs. I'm one of the few in the lab so I have good tabs on how often it's changed.
We were thinking it may be carry-over contamination. How would that produce such intense streaks?
Kudna
#4
Posted 13 September 2004 - 05:51 PM
#5
Posted 14 September 2004 - 01:34 PM
I'm in the process of receiving new PCR components, have made up fresh solutions for extractions and use sterile tips, tubes etc everytime (always have) so we will have to be patient. Thanks. Will keep you posted.
#6
Posted 15 September 2004 - 08:18 AM
/Nina
#7
Posted 15 September 2004 - 09:48 AM
Thanks.
Kudna
#8
Posted 16 September 2004 - 08:46 PM
#9
Posted 16 September 2004 - 09:19 PM
Edited by labrat, 16 September 2004 - 09:22 PM.
#10
Posted 21 September 2004 - 09:07 AM
Hope this is helpful
Ihab
p.s. check out this PCR troubleshooting site
http://info.med.yale...d/tavi/PCR.html
- Ameya P likes this
#11
Posted 22 September 2004 - 10:24 AM
The Mg2+ concentration I'm using is 1.6mM which I haven't changed so I don't think that affected it but thank you for the suggestion, and the thank you for the troubleshooting site recommendations Ihab. Very helpful.
Kudna
#12
Posted 23 September 2004 - 12:22 AM
it seems you solved the problem, however just an additional comment:
Occationally you can run into PCR primer/amplicon designs where the amplicon can in some way prime itself, to generate concatanated products which will accumulate in number and size as the PCR cycles, hence the smear. This can depend on the exact specificity of teh reaction and hence seem to reappear quite ramdomly, especially if the priming is just borderline.
What are your sequences? Try to check if there is an internal repeat, which would allow the PCR product to act as a primer on itself.
Søren
Søren M. Echwald, MSc., Ph.D.
-----------------------------
Exiqon A/S
Bygstubben 9
DK-2950 Vedbaek
Denmark
www.ProbeLibrary.com
One Real-time PCR kit, which covers 38.565 genes
#13
Posted 23 September 2004 - 12:30 AM
#14
Posted 17 November 2004 - 03:58 AM
I think i may be having a similar problem - my pcr has randomly started appearing as smears. If this was due to an internal repeat in the amplicon is there anything you could do to prevent it?
Thanks
M
#15
Posted 28 November 2004 - 07:26 PM
I think, you should redesign your primer. It should specific that amplify the target gene only. otherway, it will produce primer dimer. However, before do taht, try to increase the annealing Tm.
Also tagged with one or more of these keywords: pcr, smearing, gel
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