Need help for PCR - (Apr/06/2012 )
Can anyone please help me out regarding PCR. I don't know the reason for the non-amplification of my DNA
I am posting the protocol which I have used for PCR
Extraction of yeast genomic DNA
- Inoculated 5ml YPD aliquots with X-33 competent cells and incubated at 30oC, 180 rpm and allowed them to grow for more than 36 hours (approx 40 hours)
- 1.5 mL of culture was taken in a microfuge tube and centrifuged @ 14000g for 2 min
- Discarded supernatant
- Resuspended the pellet in 0.2 mL of extraction buffer (1% SDS, 100mM NaCl, 1mM EDTA, 2% Triton X-100 and 10mM Tris-HCl (pH=8.0) and 0.2mL of Phenol: Chloroform: Isoamyl alcohol (25:24:1)
- Cells were disrupted by adding the acid-washed glass beads and vortexed @ high speed for 3 to 4 min
- Added 0.2 mL of 1X TE buffer and centrifuged @ 14000g for 2 min
- Transferred top aqueous layer in to a new microfuge tube and added 2:1 volume of 100% ethanol (about 1mL) and mixed gently by inversion
- Centrifuged @ 14000rpm for 2 min and discarded the supernatant
- Resuspend the pellet in 0.4mL of TE and add 3ul of RNase A(10mg/ml)
- Incubate for 5 min at 37oC
- Added 10uL of 4M ammonium acetate and 1mL of 100% ethanol (ice cold)
- Mixed gently by inversion
- Centrifuged @ 14000rpm for 2min and discarded supernatant
- Air-dried the pellet in 50uL of TE buffer
Loaded 10uL of sample on to the agarose gel and ran at 100V
5uL of 10X PCR colony buffer
3uL of 25mM Magnesium chloride
1uL of dNTPs
0.8uL of each primer (of concentration 12.5mM)
5uL of DNA
31uL of sterile water
2uL of 25% Triton-X-100
0.3uL of Taq polymerase
PCR running conditions
Initialization step - 95oC for 1 min
Denaturation - 95oC for 30 sec
Annealing - 52oC for 1 min
Elongation - 72oC for 2 min
Final elongation - 72oC for 6 min
Hold – 4oC
After running PCR and loading the samples on to the agarose gel i found the bands which are exactly similar to that I have obtained before running PCR.
I also used different methods to isolate DNA- used 0.2% SDS, heat 95 C for 10 min, used lyticase enzyme, used glass beads homogenization. But none of them worked.
please help me out
What concentration was your genomic DNA? If you put 5 ul on gel, can you see it? If so, you're putting too much into the reaction. That's the first thing.
Also you didn't write what are you trying to amplify, how long the amplicon is. Are you sure the target sequence is present?
Do you know if your PCR works (primers, conditions) on some other DNA?
If it works on other DNA, and won't work on your sample even if you dilute it like 10x, 100x, 1000x, you can try adding some of the positive control to the sample, to see if it's not inhibiting PCR.
Actually, we are trying to transform P.pastoris with gp96 gene (wild type & 2 mutants) which was already inserted in to the plasmid pPICZ alpha C. we are using electroporation to transfer the plasmid in to P.pastoris (X-33 competent cells). we are using Zeocin for screening. At the beginning we got colonies but they did not express our protein. unfortunately we lost those cultures so had to repeat the transformation again. we are transforming the yeast cells but unable to get the colonies now. I don't the reason and we are still trying to express our protein. the PCR now we are running is nothing but the positive control.
so there is no chance of having any other positive control.
The amplicon size is 320 bases.
when i keep 5uL I can see a big band instead of a thin band which was like near to the 1.5 bp band of 1kb ladder DNA.
Thank u very much for your reply.
Still many things are unclear.
So you're transforming yeasts and trying to detect your gp96 gene, do you isolate yeast DNA or plasmid DNA from yeasts? If the first, is the gene supposed to be integrated into genome?
If you transformed the yeasts with a plasmid, then you can use this same plasmid in PCR as a positive control. Actually it's definitely the best control to have. You can dilute it to a concentration less than 0,1 ng/ul and use 1ul in PCR to test your reaction. But be carefull while handling concentrated plasmid, use filtered tips and don't splash over, you could contaminate your reagents.
But question was if the reaction setup - primers, program - was ever succesfully used before. So you'd know the problem is in template.
I don't really understand the last sentence.
I am sorry. I messed up. We have isolated yeast DNA. Yes, the gene is supposed to integrate into genome.
OK, then it's valid what I wrote before.
Use the plasmid as positive control of primers and reaction setup. In humans 100 ng of gDNA is a common starting amount for PCR of a single-copy gene, so calculating with the same number of template copies, that should be equal to 1 ng of yeast DNA.