Hi everyone,
I'm planning to sequence my PCR product but first i want to make sure if i prepared my sample to be amplified by PCR correctly.
I have added 10 μl 2x buffer (my taq ready mix)
1μl genomic DNA
1 μl Forword primer
8 μl water.
after words i cleaned up the product and ran it into agarose gel yet no bands appear.
should i have added 2μl of the primer rather than 1? is my mix for the pcr amplification correct? because i have done the PCR before using 1 μl of both primers and it worked fine.
thank you so much to anyone who answers.
It is not possible to say accurately if your primer is correct as we do not know the concentration but I think that you are confusing sequencing and pcr. To get a pcr product you will need both primers present. Then you purify the pcr product away from the primers with PEG, exo sap or gel electrophoretic,column purification and then you run the sequencing reaction with pcr product, sequencing enzyme mix and only one primer either forward or reverse in separate tubes. To clarify you do pcr with genomic dna and 2 primers and you do sequencing with pcr product and only one primer
dear chances are the reason you got bands in pcr when both primers were used is because of enzyme kinetics.. chances are only ur reverse primer is giving you results so what we get is linear amplification, now what can u do is change pmole concentration of primers and see the results like 0.5 ul of FP with 1.5 ul of RP and so.. see which is giving u results.. if your product size is below 600 bp u can cover whole amplicon in sequencing if more then do cloning and sequencing
Hi,
As Paul commented, it is difficult to suggest without exact primer concentrations. It is better to use higher concentration of primers just to get a band in gel and then reduce the concentration in steps to get better results. If ou get required band in gel with higher amount of primers, you could just send the unpurified pcr product to sequencing and mention the band size. it will cost extra for purification and sequencing but it is simple option.
Hi Delilah,
I think you are confusing two techniques, PCR and product sequencing. You first need to amplify your desired PCR product and then perform the sequencing. However, to make the PCR you must use both primers, F and R. otherwise you wont see nothing. I am absolutly sure that your problem is that. On the other hand, you always must explain the PCR components in terms of its concentration and not only in terms of the volume used in each case.
I agree with Paul suggestions. In addition, you can do sequence directly after ethanol cleaning of PCR product. its not necessary to use agarose gel electrophoresis.
Good Luck !
- Badges
- Science method