phosphorylate dprimers for pfx do not work - (Feb/01/2010 )
Hello, everyone!
Does anybody knows the answers for me:
I do my PCR with Invitrogens Pfx polymerase and get the good product (680 bp), but when I use the same primers with the phosphates (provided by Eurogentec), I get nothing. I need the phosphorylated product for cloning anyway.
So, the first question is why the primers do not work.
Another question - if abandom the phosphorylated primers, what is the best way to phosphorylate the product prior the ligation. I do blunt ligations into 5000 bp vector using Invitrogens ligase and kinase. Do I have to purify the phosphorylation reaction product before ligation?
Thank you for any help!!
lna on Feb 1 2010, 06:57 PM said:
Does anybody knows the answers for me:
I do my PCR with Invitrogens Pfx polymerase and get the good product (680 bp), but when I use the same primers with the phosphates (provided by Eurogentec), I get nothing. I need the phosphorylated product for cloning anyway.
So, the first question is why the primers do not work.
Another question - if abandom the phosphorylated primers, what is the best way to phosphorylate the product prior the ligation. I do blunt ligations into 5000 bp vector using Invitrogens ligase and kinase. Do I have to purify the phosphorylation reaction product before ligation?
Thank you for any help!!
you can phosphorylate your product using PNK. Yeah, you need to purify the product after PNK treatment.
DRN on Feb 1 2010, 04:23 PM said:
lna on Feb 1 2010, 06:57 PM said:
Does anybody knows the answers for me:
I do my PCR with Invitrogens Pfx polymerase and get the good product (680 bp), but when I use the same primers with the phosphates (provided by Eurogentec), I get nothing. I need the phosphorylated product for cloning anyway.
So, the first question is why the primers do not work.
Another question - if abandom the phosphorylated primers, what is the best way to phosphorylate the product prior the ligation. I do blunt ligations into 5000 bp vector using Invitrogens ligase and kinase. Do I have to purify the phosphorylation reaction product before ligation?
Thank you for any help!!
you can phosphorylate your product using PNK. Yeah, you need to purify the product after PNK treatment.
and why is it necessary? (I gel-purify the PCR before kinase reaction)
lna on Feb 2 2010, 05:13 PM said:
DRN on Feb 1 2010, 04:23 PM said:
lna on Feb 1 2010, 06:57 PM said:
Does anybody knows the answers for me:
I do my PCR with Invitrogens Pfx polymerase and get the good product (680 bp), but when I use the same primers with the phosphates (provided by Eurogentec), I get nothing. I need the phosphorylated product for cloning anyway.
So, the first question is why the primers do not work.
Another question - if abandom the phosphorylated primers, what is the best way to phosphorylate the product prior the ligation. I do blunt ligations into 5000 bp vector using Invitrogens ligase and kinase. Do I have to purify the phosphorylation reaction product before ligation?
Thank you for any help!!
you can phosphorylate your product using PNK. Yeah, you need to purify the product after PNK treatment.
and why is it necessary? (I gel-purify the PCR before kinase reaction)
to get rid of all residual matter (kinase) which might interfere in the downstream processing like ligation. i think its ok to proceed with PK trreatment w/o purification (u will cut down on the DNA loss
)....i did it once n it worked... 