Dephosphorylation of vector before ligation - Is it required? (Jan/26/2005 )
Hi!!
I´m new to this forum and to science too!!! I got here searching answers about dephosp. and i found the forum interesting and usefull... so we will keep on touch.
Bye!
can you please badcell explainmore more about your trick
Hi everyone, how about the CIAP from Invitrogen?
Hi slapolla
I don't really calculate the quantity of ER I add depending on the DNA used. I usually add 0.5 ul of ER and incubate for several hours (2-3 h, although sometimes I leave it o/n for convenience). This is more than enough, as your DNA would be on the ng range. Probably 1h of incubation will do it.
As for the buffer conditions, I sometimes dialyze the ligation for half an hour using spot dialysis filters from Millipore, and then I add the corresponding ER buffer and the enzyme. But I've also tried it without dialysis and it works. You can add directly the 10Xbuffer to the ligation mix, and the ER... and cross your fingers. For me this little trick really works.
Good luck
I like CIAP... you can calculate how much units you need according to your amount of DNA, but I after digestion, heat-inactivate RE then I just add 0.5 ul of CIAP, incubate 5-10 min, heat 65°C x 15 min. and ligate without purify!!
[/quote]
I like CIAP... you can calculate how much units you need according to your amount of DNA, but I after digestion, heat-inactivate RE then I just add 0.5 ul of CIAP, incubate 5-10 min, heat 65°C x 15 min. and ligate without purify!!
[/quote]
which means that at the time of ligation there are essentially three buffers in the system...the one for restriction enzymes, the one for the phosphatase, and finally the one for ligation (even though the former two would be in much less quantity due to a small amount of dna taken for ligation).
is there no problem? i have never done it like this, and would certainly like to eliminate one step of dna purification/ethanol precipitation.
- viv
Hi Badcell,
Here are some results I got from the strategy you suggested. I like your idea, but am a little confused about the results mentioned below. Please let me know what you think.
CONTROLS:
1) double digested vector alone: 25 colonies
2) double digested vector + ligase: 14 colonies
3) double digested; dephosphorylated; vector alone: 15 colonies
4) double digested; dephosphorylated; vector + ligase: 16 colonies
LIGATION:
5) double digested; dephosphorylated vector + Insert + ligase + RE digestion: 52 colonies
6) double digested vector + Insert + ligase + RE digestion: 45 colonies
7) double digested; dephosphorylated vector + Insert (3 different concentrations of insert) + ligase:13, 15, 11 colonies
8) double digested vector + Insert (3 different concentrations of insert) + ligase: 29, 28, 22 colonies
Do you think I should just use the colonies from ligations 5 & 6 for screening for colonies with my insert??
Thanks very much.
Hi slapolla
I don't really calculate the quantity of ER I add depending on the DNA used. I usually add 0.5 ul of ER and incubate for several hours (2-3 h, although sometimes I leave it o/n for convenience). This is more than enough, as your DNA would be on the ng range. Probably 1h of incubation will do it.
As for the buffer conditions, I sometimes dialyze the ligation for half an hour using spot dialysis filters from Millipore, and then I add the corresponding ER buffer and the enzyme. But I've also tried it without dialysis and it works. You can add directly the 10Xbuffer to the ligation mix, and the ER... and cross your fingers. For me this little trick really works.
Good luck
[/quote]
Hi, BadCell
I have same problem with cloning ds oligos, I road your comments, thank you I will do it, I want to clone a 60 mer synthetic oligo with sticky end in BglI/PstI sites of pACYC177, as you know if it works I should get the KanR and AmpS clonies, I would appreciate it if you let me know your comment,
Regards,
Farhad
i am digesting my vector with 2 diffrent rest enzymes(Bgl II + Mlu I) yeilding diff sticky ends. after digesting do i have to proceed to dephosphorylation or can i skip it. in theory the sticky ends generated above would prevent self-ligation right?
i want to know is dephosphorylation is absloutely essential in such cases.
thx
rajgene
Theoratically you dont need dephosphorytion. Now assume that you dd was 99%. means you have 1% of DNA still cut with one enzyme in mixture which can give you false result if you dont Dephosphorylate.
I would say it is not absloutly nesesarry you just need one colony. you may have to screen fe more colonies. These all are probabilites depending on digestion efficiency and optimum ratio of ligation. but there is no harm in giving CIAP for 10 minutes.
Message for Bad cell.
Hi "bad cell",
i have read ur suggestions and tricks....they r pretty cool..
i need your expert opinion and suggestion. i have posted my query in the same section, the topis is """"how to switch to a gene in a vector using PCR or restriction enzymes? gene is in pIVS2 vector""""
I appreciate your help/suggestion/comments.
Jhilmil