I have primers with Biotin tag. I use them in PCR for enrichment of particular amplicons. I have a feeling that the 6M Guanidine HCL recommended is not doing the best job. I also have to use the eluted DNA as a template, for a following PCR step.
try monomeric avidin from pierce, it requires mild elution compared to 6M Gu-HCl
Hi Bryan,
As per your suggestions, I did a control experiment. I used a Biotin-labeled primer to amplify a known fragment from a plasmid. This way all the amplicons are labeled and when I pull down with Streptavidin beads, I should be able to compare the products before and after pull down to get an idea about the efficiency.
These are the steps:
1. I performed a PCR reaction with one of the primers having Biotin, upto 30 cycles to have enough amplicons.
2. I purified the PCR products with by regular method using Ampure XP SPRI beads, which renders almost negligible DNA loss.
3. I used the Streptavidin pull down is the following way:
- I washed 25 micL of beads 2 times with Binding buffer.
- I added the purified PCR product (volume 50 micL) to the beads with 200 micL Binding buffer.
- Incubation was done at RT for 30 minutes using a overhead rotator, with lowest speed.
- I separated the beads on a magnetic rack and discarded the supernatent.
- To the beads, I added 50 micL 6M Guanidine HCL and mixed with pipetting.
- Incubation at RT for 5 mins and then separated the beads on a magnetic rack. Now, the supernatent was stored at -20 deg, which is supposed to have the products.
Alas! This did not work. I analyzed the before and after products on 2% Agarose gel. Also, I would recheck them on a Bio-analyzer, but the amount of amplicons should have been enough to be seen on a gel.
Although it's been a few months since you submitted your question, I think it's still worth it: http://www.ncbi.nlm.nih.gov/pubmed/?term=15274123
Best,
Matias
Hi, I have a related question on this thread. I am performing a ligation using 25 bp long biotinylated adapter and a smear DNA (from 100 bp to ~30 kb). I am using SPRI beads to get rid of the unligated adapters. I am getting huge DNA loss during this. Starting DNA is ~20 ug (due to variable size, I cant say what is the concentration), added about 100 uM adapter and then did SPRI purification. I have already tried a few versions of the protocol. I also tried using size exclusion Amicon Ultra comuns, DNA purification columns from Qiagen and Zymo, and few other companies. All lead to loss of mostly the high mol wt DNA.
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