Need help!!molecular cloning

[bsengez]

Hello everybody,
I have an expression vector about 4600 bp. I cloned my insert in it and made colony PCR. I got very good amplification, so I isolated the plasmids and digested if everything is okey. It should give one band around 360 bp, but now I have 3 bands which are 250,360 and 650. Any idea?

[blcr11]

How certain are you that you've cloned the correct insert? Can you tell from your colony pcr that the amplified fragment has the correct size, or were you only looking for positives without regard for what actually is giving the signal?

Does your enzyme have any kind of *star* activity and is your digestion condition conducive to that activity or not? If so, you could be seeing additional cutting from secondary cut-sites.

I'm assuming you can rule out the possibilty of extra sites in both the vector and the insert, otherwise your cloning should not have worked at all under normal conditions, anyway.

[dinofernando]

How are you purifying your plasmid, phenol chloroform or a kit. I use a kit and it works great very high yield and very fast.

[bsengez]

To Blcr11:
I'm sure that I have cloned my fragment, because I cloned it into a plasmid and controlled by sequencing, then cut with proper enzymes and cloned into expression vector. The vector and insert don't have any additional sites on it. By the way, the amplified fragment was correct size also. It is also not possible that I cloned any sites from plasmid other than my insert, because I have choosen the colonies with another antibiotics.
The enzymes that I used don't have any star activity with the buffers I used.
As you can see, there is no answer that why it happened.
Thanks for your help, any other idea?

[bsengez]

to dinofernando:
I'm also using kit for purifying. Mine works well too. Which kit are you using?

[dinofernando]

I'm using the Axygen system

[blcr11]

How many of your final clones did you look at and did they all have the same digestion pattern? If you only looked at one, then I would select 3-4 more for inspection to see if the first one wasn't just a fluke--God works in mysterious ways. If you only had one colony from the final ligation/transformation, then I would be suspicious that you picked up a rouge plasmid from somewhere. Maybe one of your benchmates plasmids got away from them? Unexpected recombination events do occur from time to time, but with todays strains it is getting to be a very rare event. I assume you gel purified the fragment for subcloning before ligation into the expression vector and that it was the correct size.

Sorry, I don't see anything obviously wrong with anything you've done. Check the original clone to make sure it hasn't changed somehow, otherwise, I'd just screen more expressor clones and/or repeat the ligation and write off the first one as a wierd fluke.

[bsengez]

Thanks for your help. I have screened 7 colonies and 3 have given amplification. I have cut 2 of them and gave same band patterns (260+350+650+vector). I'm lucky that the last one gave correct bands and I keep on cloning the other fragment.
It is called molecular biology, isn't it?

[Benjamin]

You can sequence your recombinant plasmid with vector primer, which is useful for checking your fagment has been inserted in a right frame.

[bsengez]

thanks for your suggestion. I normally sequence my plasmid after verifications, but I don't have any sequencing primers for this vector. By the way, I cloned it again and now I have no problems. So, I don't need to sequence it, thanks.

[dr. dugmore]

woh! its all gibberish to me,,... sorry cant help here ;(

[mary2184]

I am unable to insert my DNA in the pBluescript II plasmid. I am cloning sulf 1, used primary and secondary PCR, a gel extraction after every PCR. Performed a double digest with Not1 and Xho1, with both the plasmid and DNA. Then a ligation, transformation was done, blue and white colonies were observed, but after the single digest with AlfIII, the gel showed no positive results. I have performed the phenol-chloroform precipitation with ethanol, used 60C for annealing temperature on both PCR's. Ran double digest at 37 for an 1hr, 2 ul of EDTA was added during this time period then 25 minutes at 60C. I don't know what may be going wrong, if you need additional info on my protocol. DON'T HESTITATE, HELP