Phosphorylation with PNK - (Nov/03/2005 )
Hi
I tried to phosphorylate my oligoes using PNK. I used 5ul DNA (10pM), 1ul ATP (100mM), 1ul 10x buffer, 1ul PNK and 2ul H2O. I incubate my samples for 2h in 37°C. But I think my Phosphorylation process was failed. I repeated it again and again, but all of them were negative. What do you think about it??
Is it because of ATP? Formerly I was using ATP (100mM) from which was prepared as a solution by roche! My samples got phosphorylated those time, but since I changed my source of ATP all of my phosphorylations failed! my new ATP is as powder, and I make 100mM solution by dissolving it in water? Is it the reason?
I tried to phosphorylate my oligoes using PNK. I used 5ul DNA (10pM), 1ul ATP (100mM), 1ul 10x buffer, 1ul PNK and 2ul H2O. I incubate my samples for 2h in 37°C. But I think my Phosphorylation process was failed. I repeated it again and again, but all of them were negative. What do you think about it??
Is it because of ATP? Formerly I was using ATP (100mM) from which was prepared as a solution by roche! My samples got phosphorylated those time, but since I changed my source of ATP all of my phosphorylations failed! my new ATP is as powder, and I make 100mM solution by dissolving it in water? Is it the reason?
Did you check out NEB-page on that?
sure it's ATP? i once used dATP instead
how do you check your phoshorylation? radioactivity? Ya, It's really ATP, but i'm not sure if my method is tru? Can I make ATP Solution (100mM) only by dissolving ATP powder in water?
My PNK source is from Roche! What do you mean by checking NEB page? which page do you mean?
I use my phosphorylated oligoes for cloning, it's not for labelling.
this web page :
http://www.neb.com/nebecomm/products/faqproductM0201.asp#27
hi,
If you did not order your oligos with a 5' phosphate, do the following after annealing:
2uL annealed oligos, 1uL 10mM ATP, 0.5uL 10XPNK Buffer, 0.5uL H20, 1uL PNK
1hour at 37C, then heat inactivate, 10min 68C. Dilute to 200uL. Use 2uL of this dilution for your ligations.
this is from previous discussion in this forum, worked fine for me.
hope this helps
nbj
Hi all
1hour at 37C, then heat inactivate, 10min 68C. Dilute to 200uL. Use 2uL of this dilution for your ligations.
this is from previous discussion in this forum, worked fine for me.
hope this helps
nbj
I am about to do a PNK reaction with my annealed oligos (and then perform a ligation with my vector dephosphorylted) , but I need some clarifications about two points
: 1/About the quantity of dsDNA (i.e. annealed oligo's) and the number of units of enzyme for the PNK reaction. Concretely, the protocol above uses 2 uL of annealed oligos, but what's the concentration of the solution ? The Promega protocol uses 10-20 units of PNK and 100 pmol of oligo (i.e. ssDNA)...
2/After your PNK reaction, do you use a DNA carrier for your precipitation ?
Thanks for sharing your experience !
(I am a new member here, so I will contribute to this forum asap
)