Hi everyone!
I´m having problems using ExoSAP-IT™ PCR Product Cleanup Reagent for PCR purification for the first time.
I´m using 5ul of PCR product with 2ul ExoSAP-IT and then two steps of incubation 50 min 37C and 15 min 80C.
The problem is I did an electrophoresis with the PCR products (1 to 5-PCR, as seen on gel image below) vs the cleaned with ExoSAP-IT (1 to 5-EXO) to physically see the cleaning but got this results:
- An inespecific band apears in the "cleaned" product, just below the interest fragment at ~400 bp.
- It does clean the primer excess though.
Has anyone had this problem?
It can be some type of contamination even though my ExoSAP-IT is brand new?
Any suggestion?
Thanks
Hello Alejandro,
I have not seen this problem before and the only things that I can think of are that the amplimer has a polymorphism somewhere near the middle. In the reannealing step of the pcr when the primer anneals then at the later cycles the product reannneals with itself and mismatched strands re-anneal to form a pcr product that has a single stranded bubble in it. For example if we call the 2 strands N and M for the 2 base mismatches then we get NN and MM and twice as much NM plus MN strands. Then either there is some nuclease contamination or unexpectedly the EXO1 itself cuts the single stranded bubble giving a shortened double stranded minor product. Hopefully someone will come up with a better answer.
best wishes,
Paul
and while I am thinking of unlikely solutions I wonder if , in samples 1 and 2 exo there are 2 extra bands. You might consider that the highest band is pcr amplimer with some exo protein bound to it that has not finished cutting and that possibly thw smallest band is primer bound to exo1 that has not finished cutting. If this is possible you could take some exo sapped 1 or 2 and add 0.05%SDS to the sample. This would dissociate the dna and protein and the 2 bands would disappear. If this were the case then cutting less dna or cutting for longer or using more exo1 would all be likely to lead to a clean product.
Hi, Thank you very much for taking the time to answer me!
I tried both suggetions:
- First I treated a negative sample (B as seen in gel image below) with ExoSAP and got all dNTPs and Primer cleaned and no other band, as expected.
-Then I took the sample 2 from last experiment (2-Exo in first picture) and added 0.05% SDS, as you can see in the picture below (Sample 4) the bands disappear! so its most likely that the protein was bound to the primer.
The fist two samples (1 and 2 as seen in picture below) are nuclear genes and I didn´t had the inespecific band problem.
Sample 3 however, and all samples show in my first picture are Chloroplast regions, so the problem of the protein getting bound to the primer only occurs with cpDNA.
Do you think this bands (the ones formed by DNA + protein) would affect my sequencing reactions?
Thank you again for everything!
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