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#1 huyenpham

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Posted 31 January 2010 - 06:31 PM

I am doing sub cloning and I have some problems.
My interested gene is about 700 bp. My vector is pCMV-AN-GFP. I followed this protocol
1. Digest my interested gene with double digestion AsiSI (1micro) and MluI (1micro) in a 50 micro tube. NE buffer 3. Digest at 37oC during 4 hrs.
2. I also digest my plasmid with the same two enzymes and protocol.
3. Purification of interested gene and plasmid. Cut the gel.
4. Ligation vector: insert ratio 1:10 over night, using Ligation mix of Takara.
5. Transform the ligation to HST08 E. Coli and do the colony PCR with Taq polymerase using either
- forward primer: insert, reverse primer: vector --> expected product: 718 nu.
- forward primer : vector, reverse primer: insert --> expected product: 650 nu.
- forward primer : vector, reverse primer : vector --> expected product : 777 nu.
I only got my expected product when using forward primer insert and reverse primer vector. For the others, the results were the mixture of strange sized product.
then I amplify the E;Coli with plasmid contained my interested gene, and elute the plamid by the Miniprep kit(Quiagen). But when I sequence these plasmid with forward primer vector and reverse primer vector, I only saw the plasmid sequence, without insert inside. What happened to my insert? It is true that I picked up the plasmid contain my insert from the colony PCR with the primer insert and vector.
Please help, I am so confused.

#2 Qundo12

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Posted 07 February 2010 - 11:10 PM

I am doing sub cloning and I have some problems.
My interested gene is about 700 bp. My vector is pCMV-AN-GFP. I followed this protocol
1. Digest my interested gene with double digestion AsiSI (1micro) and MluI (1micro) in a 50 micro tube. NE buffer 3. Digest at 37oC during 4 hrs.
2. I also digest my plasmid with the same two enzymes and protocol.
3. Purification of interested gene and plasmid. Cut the gel.
4. Ligation vector: insert ratio 1:10 over night, using Ligation mix of Takara.
5. Transform the ligation to HST08 E. Coli and do the colony PCR with Taq polymerase using either
- forward primer: insert, reverse primer: vector --> expected product: 718 nu.
- forward primer : vector, reverse primer: insert --> expected product: 650 nu.
- forward primer : vector, reverse primer : vector --> expected product : 777 nu.
I only got my expected product when using forward primer insert and reverse primer vector. For the others, the results were the mixture of strange sized product.
then I amplify the E;Coli with plasmid contained my interested gene, and elute the plamid by the Miniprep kit(Quiagen). But when I sequence these plasmid with forward primer vector and reverse primer vector, I only saw the plasmid sequence, without insert inside. What happened to my insert? It is true that I picked up the plasmid contain my insert from the colony PCR with the primer insert and vector.
Please help, I am so confused.

Hi Huyen,
I have some suggestions:
1. The colony PCR is not reliable in some cases because of some reasons: the DNAs from the ligation mixture are still exist on the plate, and with the high sensitivity of PCR, you can get the wrong result with the amplification of DNA outside the cell (some unstable ligated form between the vector and the insert, for example). Also, when colonies grew with high density, you can pick one clone to screen, but then you pick another clone to use. Therefore, the most reliable one (if not mention the sequencing) is plasmid digestion with restriction enzymes: no wrong amplification, only one clone is analyzed.
2. Your question is quite confusing when you use the terms "primer insert" or "primer vector", because they are not proper English, so many people find it difficult to understand, and the less chance they gonna answer you.
3. If you need more helps (especially in Vietnamese if you need), you can send PM to me. Good luck!




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