ligation and miniprep - (Feb/22/2007 )
Hi,
I am doing subcloning recently. My vector is 10kb and the insert is 3.7kb. The vector was cut by xhol and inster was cut by salI and xhol. After cutting, the vector was dephospharylated by CIP. I used new england biolab quick ligase kit to do the ligation. but I did it O/N at 16C° . After ligation, I run the gel for the ligation mixture. Comparing with the cutted vector alone, the ligation give a higher bands (about 14kb) together with a band as the insert size. I transfomed to DH5a and got many colonies (more than 100).
Then I did minipre using Qiagene spin column method. I only got a band at 1.5kb. I think it is not the vector nor the insert. Then later I used 2-proponal to precipitate the plasmid instead of using the spin column, I got a higher band (bigger than 10kb) together with the 1.5kb bands. So i think the bigger band may stuck with the column and wasnot eluted out. My question is why I also have the 1.5kb band. also, which company's product is good for miniprep plasmid larger than 10kb?
If I continue get the 1.5kb band, and if the bigger band really is the plasmid I want , can I cut the bigger band out of the gel and purify it and do transformation again to DH5a cell? will the band keep the ability of transforming?
I have spend 2 weeks on this ligation, I did it 3 times. each time I got plenty of colonies and i did colony pcr, most of them are positive. But after miniprep, I only get the 1.5kb band. So please help me and give me some suggestion.
I am doing subcloning recently. My vector is 10kb and the insert is 3.7kb. The vector was cut by xhol and inster was cut by salI and xhol. After cutting, the vector was dephospharylated by CIP. I used new england biolab quick ligase kit to do the ligation. but I did it O/N at 16C° . After ligation, I run the gel for the ligation mixture. Comparing with the cutted vector alone, the ligation give a higher bands (about 14kb) together with a band as the insert size. I transfomed to DH5a and got many colonies (more than 100).
Then I did minipre using Qiagene spin column method. I only got a band at 1.5kb. I think it is not the vector nor the insert. Then later I used 2-proponal to precipitate the plasmid instead of using the spin column, I got a higher band (bigger than 10kb) together with the 1.5kb bands. So i think the bigger band may stuck with the column and wasnot eluted out. My question is why I also have the 1.5kb band. also, which company's product is good for miniprep plasmid larger than 10kb?
If I continue get the 1.5kb band, and if the bigger band really is the plasmid I want , can I cut the bigger band out of the gel and purify it and do transformation again to DH5a cell? will the band keep the ability of transforming?
I have spend 2 weeks on this ligation, I did it 3 times. each time I got plenty of colonies and i did colony pcr, most of them are positive. But after miniprep, I only get the 1.5kb band. So please help me and give me some suggestion.
A column that binds to DNA larger then 10kb, that is a tough one. I am not aware of any company that does. Limit of technology. If you do find a company that makes columns that binds well to DNA bigger then 10kb, do tell. The only thing that comes to mind is milk glass...umm no that is for genomic extraction.
As for you large band, I would suggest a restriction digest or a PCR to first determine if this really is your plasmid. It could be just your 10kb vector or genmic DNA contamination. No point going through a second transformation if this is nothing.
No idea what the 1.5kb bands is. I would place it into catch all bin of contaminant. Highly doubtfull that it is a plasmid. The selection marker gene would be nearly as big. So you could transform directly into cells.
I regularly do minipreps of different vectors with sizes ranging between 10-16 kb, get results I want. I use Qiagen miniprep kit. I personally am no expert, but I think the main problem would not be binding of DNA larger than 10 kb, but getting the larger DNA back off again (which is why I use heated tris to for elution). On a sidenote, I've also gel extracted > 10 kb with standard kits (Qiagen gel extraction and Invitrogen SNAP kits) and got DNA off (yields were ok, I also used heated tris for elution).
thanks for the info. Qiagen must have updated its kits, the ones I have say maximum 10kb...hmmm.
Update about my experiment:
Thank you for your information. I did digestion, and found the bigger band was indeed the plasmid that I want. Just as I said, I did colony pcr for them and most of them are positive.
previously, I used the column to mini prep my vector which is 10kb, it worked very well, so I just didn't pre-heat the water before I elute the ligated plasmid out. Next time I will try it to see whether the 14kb band will come off from the column if I pre-heat the water.
Also , I will try other e coli cells.
The ones I have also state maximum of 10 kb, but it works for larger ones as well.
For miniprep, I use the Macherey-nagel nucleospin kit, which gives low yield for large plasmids (~20 kb). When I do a midiprep with the Qiagen kit with the same vector, and use the low-copy protocol (100 ml per column), I get a 'normal' yield (1-2 ug/ul). I don't heat up the elution buffer or something. The minipreps of he large vectors I use for checking the sequence with RE, and if I want to do more with my plasmid, I do a midiprep.
Good luck!