Cloning into pGBKT7 vector - (Nov/25/2008 )
Hi all,
I need to subclone a 105 bp insert into the pGBKT7 vector. the vector is 7.3 Kb. I have digested the insert and the vector with EcoR1 and BamHI enzymes along with the controls. controls which i mean is the vectors cut with only one enzyme Eco or Bam.
then I extracted it from the gel and ligated in 1:3 vector insert ratio. I transformed through electroporation. I had colonies in positive control. here I used pGBK vector cut with BamH1 alone-religated and used as positive control. I also have added vector alone for self ligatiion control. I have got colonies in positive control, but none in self ligation or the ligation with vector and insert.
If you give suggesstions or ideas it would be great!
-Thilsam-
QUOTE (Thilsam @ Nov 25 2008, 06:22 PM)
Hi all,
I need to subclone a 105 bp insert into the pGBKT7 vector. the vector is 7.3 Kb. I have digested the insert and the vector with EcoR1 and BamHI enzymes along with the controls. controls which i mean is the vectors cut with only one enzyme Eco or Bam.
then I extracted it from the gel and ligated in 1:3 vector insert ratio. I transformed through electroporation. I had colonies in positive control. here I used pGBK vector cut with BamH1 alone-religated and used as positive control. I also have added vector alone for self ligatiion control. I have got colonies in positive control, but none in self ligation or the ligation with vector and insert.
If you give suggesstions or ideas it would be great!
I need to subclone a 105 bp insert into the pGBKT7 vector. the vector is 7.3 Kb. I have digested the insert and the vector with EcoR1 and BamHI enzymes along with the controls. controls which i mean is the vectors cut with only one enzyme Eco or Bam.
then I extracted it from the gel and ligated in 1:3 vector insert ratio. I transformed through electroporation. I had colonies in positive control. here I used pGBK vector cut with BamH1 alone-religated and used as positive control. I also have added vector alone for self ligatiion control. I have got colonies in positive control, but none in self ligation or the ligation with vector and insert.
If you give suggesstions or ideas it would be great!
For a 7.3 kb vector, I suggest that you use about 150-200 ng of digested vector DNA.
Which means for a 1:1 ratio you will need 2-3 ng of insert. But use about 1:5 to 1:10 ratio (10-30 ng).
You may get insert multimers, so definitely sequence the positive clones, irrespective of digestion confirmation.
-cellcounter-
QUOTE (cellcounter @ Nov 25 2008, 11:27 PM)
QUOTE (Thilsam @ Nov 25 2008, 06:22 PM)
Hi all,
I need to subclone a 105 bp insert into the pGBKT7 vector. the vector is 7.3 Kb. I have digested the insert and the vector with EcoR1 and BamHI enzymes along with the controls. controls which i mean is the vectors cut with only one enzyme Eco or Bam.
then I extracted it from the gel and ligated in 1:3 vector insert ratio. I transformed through electroporation. I had colonies in positive control. here I used pGBK vector cut with BamH1 alone-religated and used as positive control. I also have added vector alone for self ligatiion control. I have got colonies in positive control, but none in self ligation or the ligation with vector and insert.
If you give suggesstions or ideas it would be great!
I need to subclone a 105 bp insert into the pGBKT7 vector. the vector is 7.3 Kb. I have digested the insert and the vector with EcoR1 and BamHI enzymes along with the controls. controls which i mean is the vectors cut with only one enzyme Eco or Bam.
then I extracted it from the gel and ligated in 1:3 vector insert ratio. I transformed through electroporation. I had colonies in positive control. here I used pGBK vector cut with BamH1 alone-religated and used as positive control. I also have added vector alone for self ligatiion control. I have got colonies in positive control, but none in self ligation or the ligation with vector and insert.
If you give suggesstions or ideas it would be great!
For a 7.3 kb vector, I suggest that you use about 150-200 ng of digested vector DNA.
Which means for a 1:1 ratio you will need 2-3 ng of insert. But use about 1:5 to 1:10 ratio (10-30 ng).
You may get insert multimers, so definitely sequence the positive clones, irrespective of digestion confirmation.
Thank you for the suggestions.
I was trying with 6ng of insert for 100ng of vector. Its worth to try your idea.
-Thilsam-
QUOTE (Thilsam @ Nov 26 2008, 03:45 PM)
QUOTE (cellcounter @ Nov 25 2008, 11:27 PM)
QUOTE (Thilsam @ Nov 25 2008, 06:22 PM)
Hi all,
I need to subclone a 105 bp insert into the pGBKT7 vector. the vector is 7.3 Kb. I have digested the insert and the vector with EcoR1 and BamHI enzymes along with the controls. controls which i mean is the vectors cut with only one enzyme Eco or Bam.
then I extracted it from the gel and ligated in 1:3 vector insert ratio. I transformed through electroporation. I had colonies in positive control. here I used pGBK vector cut with BamH1 alone-religated and used as positive control. I also have added vector alone for self ligatiion control. I have got colonies in positive control, but none in self ligation or the ligation with vector and insert.
If you give suggesstions or ideas it would be great!
I need to subclone a 105 bp insert into the pGBKT7 vector. the vector is 7.3 Kb. I have digested the insert and the vector with EcoR1 and BamHI enzymes along with the controls. controls which i mean is the vectors cut with only one enzyme Eco or Bam.
then I extracted it from the gel and ligated in 1:3 vector insert ratio. I transformed through electroporation. I had colonies in positive control. here I used pGBK vector cut with BamH1 alone-religated and used as positive control. I also have added vector alone for self ligatiion control. I have got colonies in positive control, but none in self ligation or the ligation with vector and insert.
If you give suggesstions or ideas it would be great!
For a 7.3 kb vector, I suggest that you use about 150-200 ng of digested vector DNA.
Which means for a 1:1 ratio you will need 2-3 ng of insert. But use about 1:5 to 1:10 ratio (10-30 ng).
You may get insert multimers, so definitely sequence the positive clones, irrespective of digestion confirmation.
Thank you for the suggestions.
I was trying with 6ng of insert for 100ng of vector. Its worth to try your idea.
Even after trying the vector insert ratio differently i couldn't manage to get any transformant.
Now my question is whether i need to do sequential digestion of vector?
Also I can see a band exactly at the position of digested DNA. I couldnot see a band in the uncut slightly lower than the digested....which means that my vector may wrong or a problem in the miniprep... I dont know........
Please help!
-Thilsam-
QUOTE (Thilsam @ Nov 27 2008, 03:28 PM)
QUOTE (Thilsam @ Nov 26 2008, 03:45 PM)
QUOTE (cellcounter @ Nov 25 2008, 11:27 PM)
QUOTE (Thilsam @ Nov 25 2008, 06:22 PM)
Hi all,
I need to subclone a 105 bp insert into the pGBKT7 vector. the vector is 7.3 Kb. I have digested the insert and the vector with EcoR1 and BamHI enzymes along with the controls. controls which i mean is the vectors cut with only one enzyme Eco or Bam.
then I extracted it from the gel and ligated in 1:3 vector insert ratio. I transformed through electroporation. I had colonies in positive control. here I used pGBK vector cut with BamH1 alone-religated and used as positive control. I also have added vector alone for self ligatiion control. I have got colonies in positive control, but none in self ligation or the ligation with vector and insert.
If you give suggesstions or ideas it would be great!
I need to subclone a 105 bp insert into the pGBKT7 vector. the vector is 7.3 Kb. I have digested the insert and the vector with EcoR1 and BamHI enzymes along with the controls. controls which i mean is the vectors cut with only one enzyme Eco or Bam.
then I extracted it from the gel and ligated in 1:3 vector insert ratio. I transformed through electroporation. I had colonies in positive control. here I used pGBK vector cut with BamH1 alone-religated and used as positive control. I also have added vector alone for self ligatiion control. I have got colonies in positive control, but none in self ligation or the ligation with vector and insert.
If you give suggesstions or ideas it would be great!
For a 7.3 kb vector, I suggest that you use about 150-200 ng of digested vector DNA.
Which means for a 1:1 ratio you will need 2-3 ng of insert. But use about 1:5 to 1:10 ratio (10-30 ng).
You may get insert multimers, so definitely sequence the positive clones, irrespective of digestion confirmation.
Thank you for the suggestions.
I was trying with 6ng of insert for 100ng of vector. Its worth to try your idea.
Even after trying the vector insert ratio differently i couldn't manage to get any transformant.
Now my question is whether i need to do sequential digestion of vector?
Also I can see a band exactly at the position of digested DNA. I couldnot see a band in the uncut slightly lower than the digested....which means that my vector may wrong or a problem in the miniprep... I dont know........
Please help!
with 105bp insert in 7.5 kb, you will not see any difference between uncut clone and vector OR cut clone and vector. You need to
1. Use a newly added restriction site
2. double digest a whole lot of clone DNA to see 105 bp fragment
3. Sequence.
Sequential digest is better.
If you are not sure of complete dd, you may want to dephosphorylate the cut vector before using for ligation.
A lot of controls at all steps should help you in identifying the troublespot.
-cellcounter-