Ligation and Transformation Problem - (Jan/08/2008 )
Hello there,
This forum is awsome!! My previous question was answered quickly, but here comes another!!
I put a 1.5kb DNA piece in pcDNA3.1+ plasmid with no problem, but when I tried to put another 1.5kb piece in front of it, I got no colonies. Here is what I do, I PCR the piece, double digest the PCR product and the plasmid by double digestion-BamH1 and Hind3, 16C O/N. Ligation is performed at RT with T4 ligase O/N. However, I got no colonies~~
The ratio of the DNA to Plasmid is around 3/1, the total amount I use for transformation is 5ul/50ng DNA.
-Julietpana-
QUOTE (Julietpana @ Jan 8 2008, 07:08 AM)
Hello there,
This forum is awsome!! My previous question was answered quickly, but here comes another!!
I put a 1.5kb DNA piece in pcDNA3.1+ plasmid with no problem, but when I tried to put another 1.5kb piece in front of it, I got no colonies. Here is what I do, I PCR the piece, double digest the PCR product and the plasmid by double digestion-BamH1 and Hind3, 16C O/N. Ligation is performed at RT with T4 ligase O/N. However, I got no colonies~~
The ratio of the DNA to Plasmid is around 3/1, the total amount I use for transformation is 5ul/50ng DNA.
This forum is awsome!! My previous question was answered quickly, but here comes another!!
I put a 1.5kb DNA piece in pcDNA3.1+ plasmid with no problem, but when I tried to put another 1.5kb piece in front of it, I got no colonies. Here is what I do, I PCR the piece, double digest the PCR product and the plasmid by double digestion-BamH1 and Hind3, 16C O/N. Ligation is performed at RT with T4 ligase O/N. However, I got no colonies~~
The ratio of the DNA to Plasmid is around 3/1, the total amount I use for transformation is 5ul/50ng DNA.
1. Ligation cranking up ( positive control?)
2. Transformation not working ( positive control?)
3. seems like heat shock right here. mind letting us know how you do it?
4. try electroporation.10fold efficiency= higher chance of getting
5. plasmid should be treated phosphatase if you haven't .
6. when you say ratio issit molarity(mol) or weight(ng)
thanks
-Hanming86-
QUOTE (Hanming86 @ Jan 9 2008, 05:54 AM)
QUOTE (Julietpana @ Jan 8 2008, 07:08 AM)
Hello there,
This forum is awsome!! My previous question was answered quickly, but here comes another!!
I put a 1.5kb DNA piece in pcDNA3.1+ plasmid with no problem, but when I tried to put another 1.5kb piece in front of it, I got no colonies. Here is what I do, I PCR the piece, double digest the PCR product and the plasmid by double digestion-BamH1 and Hind3, 16C O/N. Ligation is performed at RT with T4 ligase O/N. However, I got no colonies~~
The ratio of the DNA to Plasmid is around 3/1, the total amount I use for transformation is 5ul/50ng DNA.
This forum is awsome!! My previous question was answered quickly, but here comes another!!
I put a 1.5kb DNA piece in pcDNA3.1+ plasmid with no problem, but when I tried to put another 1.5kb piece in front of it, I got no colonies. Here is what I do, I PCR the piece, double digest the PCR product and the plasmid by double digestion-BamH1 and Hind3, 16C O/N. Ligation is performed at RT with T4 ligase O/N. However, I got no colonies~~
The ratio of the DNA to Plasmid is around 3/1, the total amount I use for transformation is 5ul/50ng DNA.
1. Ligation cranking up ( positive control?)
2. Transformation not working ( positive control?)
3. seems like heat shock right here. mind letting us know how you do it?
4. try electroporation.10fold efficiency= higher chance of getting
5. plasmid should be treated phosphatase if you haven't .
6. when you say ratio issit molarity(mol) or weight(ng)
thanks
ANd need more info on the first piece of your DNA. restriction sites speaking.and what it produces maybe.
-Hanming86-