cloning help - (Aug/09/2009 )
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
yesandno on Aug 10 2009, 09:31 AM said:
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
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?
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
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.
perneseblue on Aug 16 2009, 02:06 AM said:
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
yesandno on Aug 17 2009, 12:06 PM said:
perneseblue on Aug 16 2009, 02:06 AM said:
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.
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
Stupid question: Did you check your loxP sequences (I assume that you use oligos)?
mextorquens on Aug 21 2009, 06:26 AM said:
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.