9 replies to this topic

[yesandno]

HI everybody


I am new to this site and I hope to get some help from you guys.
I have been trying to construct a vector for months. I am currently facing some weird problems that I cannot solve or explain!!

So what I am aiming to do is to clone a 3kb DNA into a vector and then insert a loxp site into the vector construct.

Things that I have done:
1. PCR the DNA and inserted it into the vector
2. sequencing result is good

then is the weird thing:
I cut the vector+insert with a RE (BlpI) and try to clone the loxp in.
colonies are grown on Amp resistant agrose plate.
colonies ratios of ligation plate and control plate is ~8:1 (not bad).
then I did miniprep with Qiaprep spin column to isolate DNA

I did double digestion to check the loxp site. The two restriction sites are on each side of the loxp (~400bp)
no DNA from miniprep can be cut!!!  ( but I can see DNA on the gel as a clear band, which is about the right size of vector+insert).
I ran a control of test digestion with untreated vector+insert (the sample that I sent for sequencing before). the double digestion worked well.

I sent a few for sequencing, the results are all negative.

I also tried to PCR the loxp region (~300bp). It only worked for the untreated vector+insert, but not the DNA from miniprep (no amplification at all).

I repeat this twice and it all give me the same result.
I wonder if those are just garbage DNA, where they came from and how to solve the problem
Plz help, I am really frustrating and depressed.


Bill

Edited by yesandno, 09 August 2009 - 08:20 PM.

[tantao]

yesandno, on Aug 10 2009, 09:31 AM, said:

HI everybody


I am new to this site and I hope to get some help from you guys.
I have been trying to construct a vector for months. I am currently facing some weird problems that I cannot solve or explain!!

So what I am aiming to do is to clone a 3kb DNA into a vector and then insert a loxp site into the vector construct.

Things that I have done:
1. PCR the DNA and inserted it into the vector
2. sequencing result is good

then is the weird thing:
I cut the vector+insert with a RE (BlpI) and try to clone the loxp in.
colonies are grown on Amp resistant agrose plate.
colonies ratios of ligation plate and control plate is ~8:1 (not bad).
then I did miniprep with Qiaprep spin column to isolate DNA

I did double digestion to check the loxp site. The two restriction sites are on each side of the loxp (~400bp)
no DNA from miniprep can be cut!!!  ( but I can see DNA on the gel as a clear band, which is about the right size of vector+insert).
I ran a control of test digestion with untreated vector+insert (the sample that I sent for sequencing before). the double digestion worked well.

I sent a few for sequencing, the results are all negative.

I also tried to PCR the loxp region (~300bp). It only worked for the untreated vector+insert, but not the DNA from miniprep (no amplification at all).

I repeat this twice and it all give me the same result.
I wonder if those are just garbage DNA, where they came from and how to solve the problem
Plz help, I am really frustrating and depressed.


Bill

I am also this new one in this field. Now I can not help you, but I think we can discuss about them
good luck!
   Tao

[perneseblue]

It is not clear to me, but I am taking that you are first ligating an insert (which has been done successfully) and a ligation of a 400bp LoxP site (that is quite big. Minimal LoxP site is only 35bp long)

as far as I can see the loxP site has failed to clone into the vector+insert molecule.

The failure of the BlpI restriction digest may indicate this. You should try another restriction enzyme to make sure, I am unsure how well does BlpI cut.

Are you PCRing the ligation mix DNA? I don't think that is a good indication for ligation success. (Are you also conducting the PCR across the junction between the vector and the LoxP site?

[yesandno]

Hi,

Thank you all for responding. I will try to make my post more clear.

Yes, I have successfully insert 3kb DNA into the Vector.  Lets call it untreated vector +3kb for now.
Then I took the untreated vector+3kb and cut it with BlpI to clone loxp into this vector+3kb construct. loxp is about 40bps. Because there is no restriction site within the loxp sequence, I have to find one restriction site on each site of the BlpI (where loxp is inserted). The distance between the two restriction sites are ~400bp. I am hoping to see a difference of 400bp (w/t loxp) and 440bp (with loxp) bands on a 2% gel. Those two restriction sites are on the vector+3kb construct, not within the loxp sequence. so even if loxp is not there, I should still see the 400bp band.

Tha same for the PCR. I was trying to PCR the loxp region. I made a PCR product length of 300bp so that I can see it on the gel. If loxp is there, I would see a 340bp band. If not, I would only see a 300bp band.

I think right now it is not the problem of Loxp. After cutting the untreated vector+3kb and trying to insert lxop, the vector+3kb (with or w/t loxp) from miniprep cannot be cut by those two enzymes that I chose to do the test digestion. The same for PCR, they cannot be amplified. Only the untreated vector+3kb construct is still working. They can be either cut by the two enzymes or be PCRed. I wonder if there is contamination somewhere so that the DNA from miniprep are just garbage DNA. But I am not really sure where the contamination is coming from.

Thanks again, guys.

Bill

Edited by yesandno, 11 August 2009 - 11:34 AM.

[perneseblue]

you could try cleaning up the miniprep with phenol chloroform. That would remove any contaminant.

Aside from  that, try cutting your plasmid with another restriction enzyme.

[yesandno]

perneseblue, on Aug 16 2009, 02:06 AM, said:

you could try cleaning up the miniprep with phenol chloroform. That would remove any contaminant.

Aside from  that, try cutting your plasmid with another restriction enzyme.

Thanks, I will try to clean my miniprep with phenol chloroform. But If there is a DNA contamination in the miniprep (from the pipette or during the process of cloning), it is not gonna slove the problem, isn't it? Like I said, the DNA from the miniprep may be just garbage DNA, so that they could not be cut or PCRed. But, anyways, I will try to do that first and see what is gonna happen. Really appreciate your suggestion. Thanks a lot.


Bill

Edited by yesandno, 17 August 2009 - 12:09 PM.

[perneseblue]

yesandno, on Aug 17 2009, 12:06 PM, said:

perneseblue, on Aug 16 2009, 02:06 AM, said:

you could try cleaning up the miniprep with phenol chloroform. That would remove any contaminant.

Aside from  that, try cutting your plasmid with another restriction enzyme.

Thanks, I will try to clean my miniprep with phenol chloroform. But If there is a DNA contamination in the miniprep (from the pipette or during the process of cloning), it is not gonna slove the problem, isn't it? Like I said, the DNA from the miniprep may be just garbage DNA, so that they could not be cut or PCRed. But, anyways, I will try to do that first and see what is gonna happen. Really appreciate your suggestion. Thanks a lot.


Bill

Yes, you are quite right. But at least you will know that the DNA's failure to cut isn't due to inhibition from contaminants.

[yesandno]

updates:

I bleached all my pipettes for 1hr. started from gylcerol stock of vecotr+3kb.
I did test digestion on vector+3kb miniprep with kpnI. The result shows correct bands.
Then I cut vector+3kb with BlpI and dephosphorylated.
Annealed new loxp, and phosphorylated it.
Tried to insert loxp with vector+3kb again.
Got colonies from control and 1:3 ligation plates.
Picked 2 of colonies from each plate, including control plate which theoretically should be shelf ligated vector+3kb.
Did miniprep again. Test digest with KpnI.
Non of the picked colonies worked. The DNA from miniprep still cannot be cut.

I dont think the problem is the miniprep, cuz I regrew vector+3kb from glycerol stock and the miniprep worked there.
I am really running of idea.
Plz help.

If I start all over, my boss would kill me. It took me half a year to get to this step

Bill

Edited by yesandno, 19 August 2009 - 12:24 PM.

[mextorquens]

Stupid question: Did you check your loxP sequences (I assume that you use oligos)?

[yesandno]

mextorquens, on Aug 21 2009, 06:26 AM, said:

Stupid question: Did you check your loxP sequences (I assume that you use oligos)?

Hi
Yes, I checked.
I even picked colonies from control plate which should be just self ligation.
Even miniprep from those colonies cannot be cut.
Since I didnt add any loxp to those vector+3kb contruct when doing ligation for the control plate, loxp should not be the problem.
I am trying maxiprep now, and hopefully the cleaner DNA would work.

Thanks.

Edited by yesandno, 21 August 2009 - 07:23 AM.