Hey,
I will clone a miRNA into the expression vector. I have 2 primer sets to get full lenght pre-miRNA, as it is shown belove;
Set 1: 5' _____________3'
Set 2: 3' ___________5'
These 2 primers have overhanging 15 nt region
After I hybridize these 2 oligos, I will run 1 cycle of PCR and get full lengt miRNA precursor as it is shown belove;
Set 1: 5' ____________________________3'
Set 2: 3' ____________________________5'
my question is that,
Each set of primers are ~75 nt . Do I really need to use page purification primers? Or can I use general purified primers. Each set requires 20 dollars, I need to clone many miRNAs, each miRNA requires 40 dollars. So this way is really expensive. Can I use only general purification?
Your time costs more than the nucleotides. $40 is pretty cheap.
But, you could always compare them. Order a few sets of primers with page purification and with general purification and see if there is any difference in the final product.
well, If you are living in a country where 20 dollar makes nearlly 100 TL, price can be a matter :)
It depends who makes the primers for sure. If you want to save money, you can PAGE them yourself, but you need to have the equipment, and if you haven't done it before, you could lose a lot of your oligo. If the sequence needs to be perfect, PAGE will not ensure there sequence is correct, only that it has gotten rid of N-1... DNA products.
There are 2 ways to go... Get them PAGE purified and you will probably need to do far less Sanger sequencing of your clone to get the correct product. If you do not get them PAGE'd, chances are you'll spend more in Sanger costs finding the clone with the sequence you want.
I've done experiments similar to these dozens of times, and I tend to PAGE anything over 60 bases. But certain vendors you might be able to get away without PAGEing. Not really an answer to your question, but observations of making genes from oligos many, many times.
Good luck
you should contact tech support at the manufacturing company, Oligos are made with cyanoethyl chemistry where the full length oligo only has a highly charged dimethoxytrityl group on it 5'end. All fail sequences and depurinated failures do not have this charged group and it can be used for purification. The problem is that if the efficiency of binding each base is 99% and the oligo is 75mer then the yield is only .99exp75 or about 47%. Fortunately these days the efficiency is much better than this but fail sequences are common and ,to some extent,sequence related. Anyway if the DMT group is left on the last base then it can be used to bind to an ion exchange column in house , the fail sequences are then washed off and then the bound purified sequence is then eluted and the DMT group removed with acid and the remaining OH group changed to an NH2 grout with ammonia. If this is done routinely or offered as a cheaper alternative by your company I think this will be a good purification method but PAGE and HPLC are better. If you were thinking of doing PAGE yourself @jack pollard on this site wrote an excellent and detailed methodology for doing it
Dear Putland,
I really appreciated for you advices. Company offered me to try MOPC colon instead of PAGE. I will try it with one primer set if it works good, than I will buy both of them the same way. If not I will buy PAGE purification. Thanks for your advices again.
Best Regards
PAGE purified primers are only needed when the normal primers cannot amplify sequences optimally. I think you should check your pcr with one set of primers and depending on your amplification decide to use the PAGE purified ones.
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