Help: Question on PCR Cleaning - PCR Cleaning Problem (Aug/02/2005 )
I have a serious problem after cleaning the PCR Product using the Omega Bio-tech PCR Cleaning Kit. First of all, the product that I get (means the DNA) is less than I thought it would be. There is another problem, I amplify about 2400 bp of plant DNA, after cleaning using the kit, I only get about half the size of DNA, around 1200-1300 bp (I use the ladder such as lambda B, 1 kb, 100 bp). This suggest that I get a single strand DNA instead of Double Strand DNA. I repeat the same experiment again and it just give me the same result. So is there anyone of you able to answer my question, and I really appreciate that. Thank you very much. Below is the Cleaning procedure that I follow:
1. Determine volume of PCR reaction and transfer an equal volume of Buffer XP1, vortex thoroughly to mix.
2. Apply the sample to a HiBind DNA spin Column and centrifuge for 1 min at 10,000 g.
3. Discard the liquid and wash the column by adding 700 micro liter of SPW Buffer (SPW Buffer dilute with 20 ml absolute ethanol), and centrifuge for 1 min at 10,000 g.
4. Discard the liquid and repeat step 3.
5. Discard the liquid and centrifuge the empty column for 1 min at 10,000 g.
6. Then add 30-50 micro liter of sterile deionized water to the column matrix and centrifuge for 1 min at 10,000 g.
It sounds like you have denatured your PCR product to ssDNA. You could add salt (150mM NaCl final) and try and renature. To do this add the salt and then put your DNA sample tube to a beaker of hot water (>90C) and let the beaker and tube cool to RT over a couple of hours.
Automated DNA sequencing reagents
Thanks Daniel, I really appreciate that. By the way, which amount of salt needed to add into the DNA sample before go into the beaker that contain the hot water.
150mM NaCl final
DNA sequencing software