Hello Yogendra, maybe you find here the answer?

http://www.protocol-online.org/biology-forums/posts/17995.html

I also got same problem when working with 28s rDNA PCR product. But without reanalyzing, the unpurified PCR product was send to MACROGEN (http://dna.macrogen.com/eng/) for purification and sequencing. The results were good. Sometimes as due to dilution in buffer (pH not 8.0) or heating the sample as per the kit protocol may be the reason for denaturation of product and hence the unwanted band as per this link by Dr.Sven Dittmann http://www.protocol-online.org/biology-forums/posts/17995.html.

Santhosh, its the same link as in my posting ;-)

Yes sir....I requoted it. Forgot to mention your name. Sorry.

No problem :-)

Thank you very much for your kind reply. I will check the details and try to avoid any mistake if i am putting in to it.

Thanks again to Mr Santosh and Dr Dittmann.

I checked it. I am using Qiagen kit for gel extraction and never heat the DNA sample, therefore i dont tkink that my problem is same as indicated in the link. In my case, my PCR product is ~1299 bp and after gel extraction and reloading in the 1 % gel i am getting one more weak band around double of its size. If any help regarding this, i will be thankful to you.

Thanks

-Yogendra

I've had some experience with end point PCR optimisation for my phD studies. Other members of my lab, at the time, experienced the same problem. Before you load your sample onto the second gel post-purification, heat your sample to 90 degrees celsius for 10 seconds. It has worked before which leads us to think PCR products can become "entangled" through inter-product hydrogen bonding. Thermal denaturation would therefore abrogate this. Give it a go and let me know. Fingers crossed!

I also suggest you to incubate the sample to 70-90 degrees before applying to the gel. Your amplicon has a lot of GC %?

Yogendra, I have experienced and a few people have also mentioned it that the Buffer QG many times degrades the DNA you want to purifiy, coupled with heating. I have experienced the either the DNA is degraded or the concentration is reduced during the extraction. Either way its not very desirable if you need high concentration for downstream processing. if you are goin for sequencing you can use it. May be the same problem is happening there. Try using a different kit or Crush and Soak or electroporation methods . if these work fine then the problem may be with the kit.

I used to extract from gel, but switched to the PureLink PCR Micro Kit by Invitrogen. This is the ONLY kit (I tested five other kits by various manufacturers) that does not retain primers and cleans the PCR product efficiently enough to use in a very sensitive quantitative real-time PCR protocol. You avoid a number of problems this way, but I won't go into this. Just make absolutely sure to use this kit only.

Thanks to all for their kind reply. I think i should make my case a bit more clear becoz all the suggestions seems to be inapplicable in my case. Here it is..... What i am doing is that i am amplifying a 1300 bp gene from Vibrio sp. which has 48 % GC content. When i am doing PCR, i am able to get a very good amplification with one additional band at below 3 kb size which is actually very weak. So i thought that i must purify the PCR product before directly going to clone it. When i purified it with gel extraction method, upon reanalyzing on agarose gel i found another band at just below 3kb. So i again extracted my 1300 bp gene as single band and purified it. To just check the purity of this, i again ran the gel but the problem was same. i am still getting the higher MW band. Now, becoz it is higher MW so we can not say that my sample prep is suffering with degradation. I used Qiagen kit for this all. And the very strange problem ahead is that when i tried to clone it in some easy vector like pGEM-T to make my further cloning easy, i got many false white colonies. When i screened the white colonies i am getting many different inserts of different size. How it can be when i am using only 1300 bp insert? Then i ruled out the possibility of multiple occurrence of a cutting site by digesting it with individual cutting enzymes at a time. Here i got only single bands every time. But the moment i am applying double digestion, the resulting insert show different bands in different white colonies. Now i am planning to do sequencing at this step with the colony which giving me atleast 1300bp sized fallout.

Now, considering these all facts can anybody suggest me how to go for cloning of this troublesome gene?

I am very sorry to right in that much detail but i think it would help us in solving the problem.

With best regards

-Yogendra

I am unable to understand that when you excise only your required band from gel how is this possible that gel purified sample contain additional band. there may be a chance that one of the solutions in gel purification kit got contaminated. you may also overcome problem of getting an additional band by using nested primers. do you prepare positive and negative control reactions while doing PCR? also check properties of primers like homo n heterodimers ...... it may also be the reason for non-specific amplification.

Good luck

also i want to add something Qiagen kit is not at giving you good results.........we use Promega Gel clean up kit and its results are wonderful

Reduce your template concentration in your initial PCR step (BEFORE PURIFICATION). High concentration of template also leads to non specific bands. Dilute the template in sterile ultra pure water. Your primary PCR product is not proper so purification may not help. Then troubleshooting annealing temperature is the second thing you can try if diluting template fails. Diluting the template will work definitely.

Yogendra, if you are looking for a quick way to recover only your desired 1300 bp PCR product, I might recommend using a size exclusion system. I have use the E-gel system from Invitrogen for fragmentation experiments, and it has worked well (+/- 100bp) for targeted size products. It is a bit more expensive that using a standard agarose gel, but you don't have to bother with a purification step after PCR or any dyes or ethidium bromide.