Difficulties cloning by PCR
Posted 12 October 2009 - 05:57 PM
I find in difficulties in cloning full length of Tobacco cDNA, about 1764bp.
PCR system I used is
10xPCR buffer 2ul
cDNA as templates 1ul
Primers I used is
Its Tm and GC % is 57C, 59C and 34%,...., respectively.
Reaction Program is as follows:
final extention 72C 10min
Here, Tm I used is between 55-65C, 58-66C, 55C.
I also tried to clone it by using Touchdown PCR.
Reagent system I used is
10x PCR buffer 5ul
cDNA as Template 1ul
Program is as follows:
TM 1 45s
72C 2min/1.5 min
Tm 2 45s
Here, Tm 1 = 57C, 58C, 60C, 62C, 63C , respectively
Tm 2 = 55C, 56C, 58C, 60C, 61C, respectively.
from last 3 months until now,
I didnt get any bands.
I hope U can solve my serious difficulties.
Thank you very much!!!!!!!
Posted 13 October 2009 - 06:58 PM
I supposse that the amount of primers, template and dNTPs are the correct ones because you talk in uL terms and it would be easier to talk in mass or molar terms. Anyway, have you rigorously checked your primers sequences? is the template nucleotide sequence the one you thought it is?
Edited by celvas, 13 October 2009 - 07:06 PM.
Posted 14 October 2009 - 12:48 AM
I would try different concentrations of primer, template and MgCl2 on a gradient PCR (which I'm assuming it is what your first protocol is) and take it from there. Also, how GC rich is your template? You might need to add some additives such as DMSO or betaine.
Do you have a positive control? Even if is different template and primers, at least to confirm that your PCR reagents actually work?
I've just seen what I assume is a typo, or me understanding it wrong but what does: "primer 1/1ul" exactly means? is that 1ul of primer 1, if so, where's primer 2? Is it 1ul of each primer, again what concentration? what do you mean by 1/1ul???
Posted 14 October 2009 - 03:50 AM
Posted 14 October 2009 - 04:05 AM
BTW, Primer3 calculates that your first primer (atggcatctttttctgttaaattctcagc) has a Tm of 65.6 °C, and your second primer (ttaatcttctgcttcttctctttctc) has a Tm of 58.2 °C -- this is a huge difference in Tm's, and might be the cause of your difficulties. If you drop four 3' bases from the first primer (thus making it a 25-mer: atggcatctttttctgttaaattct), the Tm is 58.7 °C, a much better match for your second primer.
Posted 14 October 2009 - 04:25 AM