smear in long distance PCR

[Lina Sun]

Hi,guys,
I´ve been performing a long PCR determination of RyR from one insect. cDNA was template, I could manage to amplify the corresponding bands(15.4K). In the first PCR, indistinct specific bands. Then 2nd PCR, smear bands or no bands, why?

[butters]

Is the primer identical for first and second PCR? If your first PCR is ok why do you need second PCR so we could give you better suggestion for your downstream application.

15.4kbp is pretty long. Well, how much DNA samples/template did you take from first PCR to second PCR? I somehow feel that it maybe due to too much DNA. But would only know if you provide more information. Thank you.

[Lina Sun]

In first pcr, 1 microlitre cDNA was template; and in second PCR, 1 microlitre first PCR product was template.

[butters]

I don't generally share this but I guess I may not be doing much scientific labwork soon. Lina try something for me if possible.

1. Perform first PCR as usual, load say 10uL - 20uL of PCR product depend how much you can spare for running on agarose gel. For large volume make sure that you have correct comb size for it.
**reason for this is the determine any small amount of other PCR amplicon that may present.

2. Also prepare you PCR mix without template (don't do it too long, as non specific may occur if you prepare your mix then run gel. Plan that so when you finish preparing PCR mix you got your gel run finish). Use a sterile 10uL tip, stab few times on the band that you want and "inoculate" the tip on your PCR mix. run it. I would however like to suggest lower PCR cycles but since your is long PCR just run as it.

I found this protocol somewhere in the internet, I can't remember where. So please do not refer me as the original person with this idea. I love this kind of methods to help and troubleshoot PCR.

Another thing is your PCR protocol, is it touchdown or ?...

[Lina Sun]

Thank you for your suggestion, I have tried the method that you suggeted, the 2-step PCR protocol was used, but it's a pity that I couldn't get the specific band.

[butters]

Lina, do you still see the smear? Please update us

[Lina Sun]

Now, I couldn't see the smear and specific band too. Wolud you help me? What methods will be used to get the full lengh cDNA(15372bp)?

[butters]

Is it using the method I recommend or your usual routine method? The method I suggested is to select specific amplicon to be amplify. It is helpful if you don't want to / no protocol to purify PCR amplicon. Its fast and can be done anywhere.

Ok, don't panic. Is the negative result reproducible or was it only one time? Do you frequently thaw and freeze your DNA template? Generally if you don't prepare DNA template freshly for each run, it is best that you aliquot it to few tubes. This is especially true if you template is very long.

What Taq and PCR protocol, PCR machine are you using? It will influence your result. Make sure your Taq is able to amplify long PCR product and you are using recommend duration for amplification.

Ok just in case that I go into hibernation MODE again. Check link below. It will give you some guide. Do you have senior labmate in the lab? Maybe they could help out. As I understand sometimes some information maybe too sensitive to be given out here. Are you the only person doing this long PCR or there are others?

[Only registered users see links. ]

It seem like there are alot of parameter to look at so it is best that you check with the essential first. So many parameter to look at to make the next best move. So you read the guide first then we can start one by one.

[butters]

No, we won't be testing all just the one to give you sufficient result for next step. Anyhow what is the objective of your work? Is it to amplify product only? Or inclusive of insertion into cosmid or something? So it would be easier to just focus for that.

[Lina Sun]

I want to upload PCR electrophoresis picture, but I couldn't do it, would you mind telling me your E-mail?