yasamino, on 26 August 2011 - 11:46 AM, said:
Adrian K, on 26 August 2011 - 11:02 AM, said:
There are six lanes, For band number 2 &3, there are 2 bands.
Maybe I am wrong, but I do not find that your band sizes were corresponded with the size you wanted. it is like 1.2kb and 1.8kb. Which brand of 1kb ladder you use, can provide the catalog number as well as a reference?
Let's say the band you wanted is the bottom band, I suggest you directly just pool all your samples, run it in agarose gel, do a gel purification and elution and finally do a cloning into your desired vector. There is no need to waste so much time on PCR optimization.
Is hard to say in this stage that whether your cDNA is degraded or whether you should increase it. I suggest you try to use your genomic DNA as control and see if there is any differences, provided your primer was not designed on the splicing site. Also, addition of Betaine or DMSO might help to eliminate the unspecificity binding and perhaps reduce smear. This is not the issue of primer dimmer.
Thanks for your reply. Much appreciated. I am sorry for my questions, I noticed you mentioned you work long hours in the lab. I am really sorry if I am being a bother.
The ladder I am using is a 1kb ladder from Invitrogen and its Cat # is 15615-016.
Also I do notice two bands in each lane, you are right, but I thought the second band on the bottom would be my right size, since the in the ladder the bright band is 500bp and right above it would be close to 585bp?
OK so you think gel purification is a better idea than PCR clean up using the PCR sample itself, rather than gel?
And when you say genomic DNA do you mean I order a DNA extract of my gene only and use that as a positive control with my primers?
I was thinking of using the DMSO and or formamide today but I thought I should receive some feedback from the forum
But really thanks alot, you are great help
Haha, don't don't apologize, is not your fault. When I plan my work, I forget that almost the next whole week is my country's holiday, and I can't use most of the instruments. Most of the staff will be absent, and thus the office closed.. I got no choice but to stay back for "lab marathon".
Yes, gel-excised purification is always better as it only cut out and isolate the band you wanted. Sorry for make you confuse in my previous post. The shortcut (a.k.a cheat) I was trying to say is you can use a PCR clean up spin column, load all of the remaining PCR products from your gradient PCR (would be roughly 15ul from each tube times 6 equals to around 90ul total volume), do a clean up and elute the product (using around 40ul elution buffer). After the elution, load all the eluted product onto agarose gel, run it, and gel-excised-purified the band you required (your case, cut only the 585bp band).
Alternatively, you just load all your ~90ul product onto the agarose gel, run the electrophoresis and proceed with gel-excised-purification for the 585bp band. During your elution step, try elute with less than 40ul elution buffer (30ul would be even better as it 3x concentrated your initial volume of ~90ul). Use the product for your cloning purposes.
My suggestions is mean for you to speed up your work rather than just focus on PCR optimization. Those were the few tricks I use for my work, yet to fail me for the almost 30 different clones I did. Just imagine the time you required for optimizing 30 independent PCR, unless you are using for endpoint detection.
However for your cloning purposes, as phage434 mentioned earlier, you might not get it done due to those RE required extra 5-6 bases. I suggest you do a TA / Blunt end cloning first, send for sequencing and after your new primers arrived, you PCR it again using the cloned plasmid, so that you do not need to use your cDNA again.
For the PCR addictives, I never heard of using "formamide". The addictives commonly used is betaine, DMSO and BSA.
Hope this helps.
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