The ds-dDNAs were generated by annealing 10 μg of
both forward and reverse oligonucleotides in a total volume
of 20 μl at 95 °C for 5 min and allowing the reaction
mixture to cool down at room temperature. The
annealing reaction was verified by loading 500 ng of each
single-stranded oligonucleotides and 1 μg of total DNA
in the annealing reaction and by visualizing the bands
using ATLAS ClearSight DNA Stain (BIOATLAS). The
bands corresponding to the single-stranded oligonucleotides
disappeared in the annealing reaction sample.
Could anyone please explain this content regarding the donor DNA preparation?
Is the procedure you list something you did? Or is it from a paper and you are asking how it works?
It is actually from a paper (DOI 10.1186/s12934-017-0681-1). I need to know the exact procedure for preparing donor DNA (ds-dDNA). I tried to edit the genome of Edwardsiella piscicida using the CRISPR/CAS technique for more than a year, but I failed. I followed overlap PCR for donor DNA preparation, but I didn't use the procedure mentioned in my first question. Actually, I'm totally confused with this whole study, and this is the main part of my PhD project. If you have any idea or suggestion, please let me know, and thank you very much for your kind response.
So the protocol seems very straightforward. If you have two oligonucleotides that are complementary, then they will automatically anneal with each other and form a double stranded molecule. Often we heat them first to break any intramolecular hydrogen bonds that are formed internally and then during the cooling process they form the most stable structure which is the double stranded form.
To confirm that this worked, the authors ran a gel with 3 samples, one was the control with only one oligo, the second was also a control with the second oligo, and the third was the sample with both having annealed. You should see a size difference with the third. Most likely this sample will have some bands for all 3 sizes.
Are you sure that your oligoes are correct? Or maybe the problem is some other step in the protocol?
I would like to know how to prepare the two complementary oligonucleotides. If you have any possibility, could you please explain the procedure of the preparation of the two complementary oligonucleotides?
Previously, I followed an overlap PCR technique for making donor DNA as follows.
I planned to delete 30 bp's of the selected gene. So, I identified the 30 bp protospacer with the PAM region. I produced 170bp long mutagenic donor DNA without 30 bp protospacer+PAM.
First, I designed forward primer and reverse primer for the upstream of the protospacer region. The upstream reverse primer contained a 15 bp overhang, which skipped the protospacer+ PAM region. Second, I designed primers for the downstream of the protospacer region as previously. The downstream forward primer contained a 15 bp overhang which skipped the protospacer+ PAM region.
Upstream and downstream arms were separately amplified using genomic DNA and gel purification was performed. After that, overlap PCR was performed using upstream forward primer and downstream reverse primer with previously amplified upstream and downstream arms (1:1) as templates. The amplified product was gel purified and used as donor DNA for cotransformation with the pCRISPR plasmid.
Here, I have attached a document that contains details of the primer and the selected gene that I used for this study. Please be kind enough to go through it if you have free time. Thank you very much for your kind concern.
Two complementary oligonucleotides are just the opposite strands of DNA. In your attachment, the two primers at the bottom are complementary oligos except for the sequences at the 5' end of each.
Those bottom primers were used for oligomerization, and they were used to clone pCRISPR plasmids. Could you please let me know whether the donor DNA preparation protocol I have described is correct or not?