Get stuck in a subcoloning: put 0.6kb into 7kb - (May/04/2006 )
Hi, there,
I tried very hard to put a 0.6kb fragment into 7kb vector. I created a Pac I site on vector, sequenced to make sure the Pac I is there. Then cut O/N, CIAP (NEB), cleaned twice with QIAGEN kit (in case couldn'd get rid of CIAP). And used PCR or add linker method to created Pac I site on insert. Sequenced insert. Ran gel to check the size and concentration of vector and insert. Tried different ligation ratio from 1:1 to 1:20. I used NEB T4 ligase, 16C, O/N. But, control group (vector only)'s background was alway 10-20 colonies and the experiment groups were not 2X more than control group, and all false-positive. I cultured the colonies and digest with Pac I, some colonies had single band, some even had several big size bands! But none of them had the insert.
I couldn't image what is going on now. Please give me some suggestion. Thanks.
I tried very hard to put a 0.6kb fragment into 7kb vector. I created a Pac I site on vector, sequenced to make sure the Pac I is there. Then cut O/N, CIAP (NEB), cleaned twice with QIAGEN kit (in case couldn'd get rid of CIAP). And used PCR or add linker method to created Pac I site on insert. Sequenced insert. Ran gel to check the size and concentration of vector and insert. Tried different ligation ratio from 1:1 to 1:20. I used NEB T4 ligase, 16C, O/N. But, control group (vector only)'s background was alway 10-20 colonies and the experiment groups were not 2X more than control group, and all false-positive. I cultured the colonies and digest with Pac I, some colonies had single band, some even had several big size bands! But none of them had the insert.
I couldn't image what is going on now. Please give me some suggestion. Thanks.
Hi.
If I ever get those kind of problems, Ido an insert ligation control. Take a reasonable amount of your prepared insert (with the Pac I site), and self-ligate it (This shouldn't be a problem if you're starting with PCR product). After a 1 hr ligation (this is a bit of a quick-and-dirty test, after all), run some on a gel. If your Pac I site has been made correctly, you should get a smear; if not, you have found your problem. You might get a 600bp band, or a 1200 bp band, which will tell you if the RE site expt has worked partially or not at all.
If you want to, you can digest this ligation control with Pac I. This will guarantee that all of the ends are Pac I. Use this in your vector ligation expt.
btw, how did you sequence your insert if it wasn't cloned in somewhere?
Good luck.
Seems that ur CIP is not working. Dont use more than 100 ng DNA in a total volume of 10 ul.
and ratio vector:insert from 1:1 to 1:3 should be fine (eg. for 1:1 70 ng vector 6 ng insert). Double check ciping protocol or borrow some new CIP. Are you daeactivating PacI 20 min 65 ^before ciping? maybe will help so it doesnt stick to the ends after digestion.
Good luck
I tried very hard to put a 0.6kb fragment into 7kb vector. I created a Pac I site on vector, sequenced to make sure the Pac I is there. Then cut O/N, CIAP (NEB), cleaned twice with QIAGEN kit (in case couldn'd get rid of CIAP). And used PCR or add linker method to created Pac I site on insert. Sequenced insert. Ran gel to check the size and concentration of vector and insert. Tried different ligation ratio from 1:1 to 1:20. I used NEB T4 ligase, 16C, O/N. But, control group (vector only)'s background was alway 10-20 colonies and the experiment groups were not 2X more than control group, and all false-positive. I cultured the colonies and digest with Pac I, some colonies had single band, some even had several big size bands! But none of them had the insert.
I couldn't image what is going on now. Please give me some suggestion. Thanks.
Hi, there,
Inactivate after CIAP by heating it 65C for 20- 30 min. Even if you purify with qiagen kit, there might still be some remnants.
Also limit the amount of time of digestion. Do it during the day for 1-2 hrs.
Heat the insert upto 65C for 5 min. then let it cool down for 15 min. Then try to ligate using 100ng of vector and atleast 50 ng of insert, if more even better.
Good luck !!!
I would design the experiment in such a way where you do not need to use CIP.
I would try a double digest using primers containing the RE for the insert.
-Matt
I would try a double digest using primers containing the RE for the insert.
-Matt
Thank you so much very body. I ran vector self-ligate before CIP, it worked very well. Now I will try to using 2 REs instead of one to skip CIP. Maybe that gave me problem.
Have a good weekend.
Hi, sometimes cloning can be problematic even if you do everything as it should be. Good luck with all the recomendations you got here.
Another option is ligation by recombination, here you have a reference, I think there are more:
Nucleic Acids Res. 1990 October 25; 18(20): 6069–6074.
Ligation-independent cloning of PCR products (LIC-PCR).
C Aslanidis and P J de Jong
This procedure is used these days by the Structural Genomics Consortium people, for high through put cloning and it is working great for them. You can try. I think BD biosciences sells some kit related to this procedure.
Good luck again.
M
Another option is ligation by recombination, here you have a reference, I think there are more:
Nucleic Acids Res. 1990 October 25; 18(20): 6069–6074.
Ligation-independent cloning of PCR products (LIC-PCR).
C Aslanidis and P J de Jong
This procedure is used these days by the Structural Genomics Consortium people, for high through put cloning and it is working great for them. You can try. I think BD biosciences sells some kit related to this procedure.
Good luck again.
M
Thank you very much Marcfe for your infomation. I created 2 REs, didn't dephosphotate, and i got it.
Again i appriciate you guys help.
Best
Yes, I am one of those high-throughput structural genomics-type people, and we do sometimes use it.
I still think I prefer good old RE double-digest and ligase, however. In the long-term, it's cheaper to work with RE and ligase than to order twice as many primers. Whatever works, works, though...it's always nice to have another trick up your sleave should something fail.
-Matt