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wrong bands after T-vector cloning - (Jun/26/2009 )

Hello All,
I was doing PCR cloning using pCR-TOPO2.1 vector

my PCR product is ~1kb with three RE sites (AflII, AscI and HindIII). After PCR using Ex taq, I ran the gel, eluted the band from gel and then did gel purification. After gel purification, i ran the gel again to make sure the right size and right band.

I ligated the PCR product with pCR-TOPO2.The size of the plasmid is around 5kb. 1 and got several white colonies and few blue colonies. I picked few white colonies and blue colonies, checked with different REs. The digestion time was around 8 hr.

The first REs i used are AflII and AscI and this should give me two band. One is my insert, and the other should be the vector. I got two bands with correct sizes.
The second RE has three cuts, one on the insert and two on the vector. But I only got one single clear band with 5kb. But this band didnít look like uncut DNA since there were no other two bands that indicate the uncut plasmid.
The third REs has two cuts, one on the insert and the other on the vector. However, I only got one band with strange size (~3kb).

The 2nd and 3rd results were strange. The 2nd could be due to the incompletely digestion. But Iíve digested for 8 hours. Is it possible that it is not cut completely? As for the 3rd result, I really donít know how to explain.

Should I try different REs, or should start over the cloning??

Can someone help me? Thank you!

-Ah-Do-

1, For the 2nd experiment, extra bands should show even with uncomplete digestion. Where is the supercoil plasmid band of 6kb? It may be around 5kb position or less. Put a control lane without digestion to compare. What're the expected band postions? If the enzymes can not digest the plasmid, check whether enzymes used could be blocked by methylation modification or not. Check the buffer condition. How do you cut the plasmid by three enzymes? One by one? or three together?
2, What's the product length for each in the 3rd digestion? If both are close to 3kb, they may show in one band.
3, Check the sequence of the plasmid if possible. That will give you confirmed information with less cost in both time and money.

Ah-Do on Jun 27 2009, 12:30 AM said:

The second RE has three cuts, one on the insert and two on the vector. But I only got one single clear band with 5kb. But this band didnít look like uncut DNA since there were no other two bands that indicate the uncut plasmid.
The third REs has two cuts, one on the insert and the other on the vector. However, I only got one band with strange size (~3kb).

The 2nd and 3rd results were strange. The 2nd could be due to the incompletely digestion. But Iíve digested for 8 hours. Is it possible that it is not cut completely? As for the 3rd result, I really donít know how to explain.

Should I try different REs, or should start over the cloning??

Can someone help me? Thank you!

-Zhongbo-

Zhongbo on Jun 27 2009, 01:48 PM said:

1, For the 2nd experiment, extra bands should show even with uncomplete digestion. Where is the supercoil plasmid band of 6kb? It may be around 5kb position or less. Put a control lane without digestion to compare. What're the expected band postions? If the enzymes can not digest the plasmid, check whether enzymes used could be blocked by methylation modification or not. Check the buffer condition. How do you cut the plasmid by three enzymes? One by one? or three together?
2, What's the product length for each in the 3rd digestion? If both are close to 3kb, they may show in one band.
3, Check the sequence of the plasmid if possible. That will give you confirmed information with less cost in both time and money.


maybe i didn't explain clearly that made you misunderstand my questions.

1. the 2nd experiment, i only used ONE enzyme. My plasmid has three such RE sites. so, supposedly, i should get three bands, but i only got one single clear band. the size is 5kb which is the size of my plasmid. it looks like the linearized plasmid. I was thinking, is it possible that only one RE site is valid or the other two RE sites are not working?? that's why i was thinking of using different enzyme to cut.

2. the 3rd experiment, i also used ONE enzyme that has two cuts on my plasmid. I should get two bands (~1500 kb and ~3500kb). However, i only got one band which is ~3kb. i used NEB 1kb ladder that should be able to separate 1500kb and 3500 kb bands. The 3kb band i got didn't seem to belong to any possible cut.

3. it is a cloning vector. Is it necessary to do sequencing? is sequencing a way to pick out the positive clones?

-Ah-Do-

Ah-Do on Jun 29 2009, 04:19 PM said:

Zhongbo on Jun 27 2009, 01:48 PM said:

1, For the 2nd experiment, extra bands should show even with uncomplete digestion. Where is the supercoil plasmid band of 6kb? It may be around 5kb position or less. Put a control lane without digestion to compare. What're the expected band postions? If the enzymes can not digest the plasmid, check whether enzymes used could be blocked by methylation modification or not. Check the buffer condition. How do you cut the plasmid by three enzymes? One by one? or three together?
2, What's the product length for each in the 3rd digestion? If both are close to 3kb, they may show in one band.
3, Check the sequence of the plasmid if possible. That will give you confirmed information with less cost in both time and money.


maybe i didn't explain clearly that made you misunderstand my questions.

1. the 2nd experiment, i only used ONE enzyme. My plasmid has three such RE sites. so, supposedly, i should get three bands, but i only got one single clear band. the size is 5kb which is the size of my plasmid. it looks like the linearized plasmid. I was thinking, is it possible that only one RE site is valid or the other two RE sites are not working?? that's why i was thinking of using different enzyme to cut.

2. the 3rd experiment, i also used ONE enzyme that has two cuts on my plasmid. I should get two bands (~1500 kb and ~3500kb). However, i only got one band which is ~3kb. i used NEB 1kb ladder that should be able to separate 1500kb and 3500 kb bands. The 3kb band i got didn't seem to belong to any possible cut.

3. it is a cloning vector. Is it necessary to do sequencing? is sequencing a way to pick out the positive clones?



mmm...
after the strange REs digestion resutls, i used the minipreps from pCR-TOPO-my gene plasmid to do PCR again. I used the original plasmid as the positive control, and also do a negative control (without plasmid). Surprisingly, all the pCR-TOPO-gene products gave me the same results. they all showed a band with the size of my fragment.

it is so strange. could anyone explain to me?
1. if the PCR results of the pCR-TOPO-gene are correct, that means the T-vector ligatioin worked? and my insert is in the pCR-TOPO vector? 2. then, why when i checked with different sets of enzymes, they gave me different bands with wrong sizes?

it is really confusing. please help me figure out this puzzle.
thanks a lot!

-Ah-Do-