subcloning help urgent! - (Apr/12/2007 )
Hi guys,
I have got a problem with my cloning work. First my plan is PCR my insert (1.1kb) using normal Taq----- then do TA cloning-----then select the positive clone-----then minipreps it------digest positive clone and the vector (6.3kb) I want to insert the fragment into with BamH1 both and treat the vector with CIP----then do the ligation of them.
What I achieved are that
1 I have got the TA clone finished successfully (a lot of positive clones on my plate)
2 the CIP treated vector only (without insert) has no colonies which means CIP worked.
3 the non-CIP treated vector control (only digested with BamH1) came up with lots of colonies which means ligase and buffer should be alright.
whereas
I have got no colonies on my insert and vector plate.....
I have checked the gel extracted and BanmH1 digested insert and vectors on the agarose gel. they all ok (Bands all visible with ease).
The two things I am concerning are:
1 I prepare the BamH1 digested insert for ligation about 10 days before I did the ligation with vector. I kept it in -20 and I suppose it shall be ok. But what do you guys think. Would this be a potential problem.... I mean, if you leave it longer, the phospho group can detach from the DNA backbone? and result in unsuccessful ligation due to non any phospo group there to allow ligation
2 the ratio of the ligation: On my gel, the amounts of the vector and insert seems equal, and then I used 2.5 microliter of the vector and 4.5microliter of the insert in 10 microliter total reaction volume.
Dose the ratio I used interfere with my ligation efficiency?
Can anyone give some kind help plz???
I really appreciate that!!
It could be lot of things....
- try with differents ratio of vector-insert
- for the insert store at -20°, you can try with a fresh one, but I'm not sure that it works!
- maybe you have a problem with the transfection
- are you sure about your selection (antibiotics?)
- An other point, for some cloning, UV degrade DNA so your vector or insert... Do an agarose gel with a part of the material and a other ne with the rest. Take the first one to have a idea about the position of the band an cut the second gel at this position but without UV. It's more difficult...
- check the map of your vector to be sure to have good enzyme site
It's just some suggestions, so good luck 
- try with differents ratio of vector-insert
- for the insert store at -20°, you can try with a fresh one, but I'm not sure that it works!
- maybe you have a problem with the transfection
- are you sure about your selection (antibiotics?)
- An other point, for some cloning, UV degrade DNA so your vector or insert... Do an agarose gel with a part of the material and a other ne with the rest. Take the first one to have a idea about the position of the band an cut the second gel at this position but without UV. It's more difficult...
- check the map of your vector to be sure to have good enzyme site
It's just some suggestions, so good luck
thx for your reply,
As your said I am preping new flesh vectors for digestion.
I think the transformation not transfection (as you mentioned) might not be a problem, otherwise, how to explain with the religated vector many colonies are coming out.
and I am sure of the antibiotics selection of my vectors (otherwise all the plates will appear with lots of colonies right?) but mine, just one plate not the rest two.
and if the UV causes the degradation of DNA, again the possibility should be equal, that means the one without the CIP treatment
should also have a potential to be degraded, but They can religate and be tranformed quite well. I and I did expose them for a short period and I think might not be a problem as I did the same thing on my first TA cloning and many others use the same way to do it straight. But anyway I appreciate you idea. maybe it is worth trying.
and the enzymes should be alright....
http://www.sigmaaldrich.com/catalog/search...amp;Brand=SIGMA
it is the sigma 3*FLAG-CMV-10 vector for stable expression
there is a BamH1 site there
and my insert contain BamH1 as well...
Thank a lot for your help
Hi
in the past I have found problems ligating gel purified fragments with dephos vectors. I have resolved this by treating the gel purified fragment with polynucleotide kinase (this can be done in ligase buffer) or preferably not performing a gel purification of the insert and just setting up the ligation with a 1:1 ratio (instead of 1:3) so that there is greater emphasis on the receptor vector than usual, then digesting the donor vector away after ligation (ie cut with something not in the gene or in receptor vector, but that will linearise any re-annealed donor vector.
Both of these methods have been succesful on a number of occassions for me.
good luck
jenjon
CIP can be difficult to inactivate try SAP or do a phenol extraction to make sure CIP is gone.
Hi Beccaf22, thx for your reply, but I don't somewhat get that. Did your mean, CIP can interfere with the later ligation? I know when you run the CIP treated samples on the gel, You inactivate CIP by add the DNA loading buffer, is that right? and even it dose not help, whn you run DNA on the gel, the CIP will be gone into the TAE buffer (no charge on it, so is not subjet to electrophoresis) ...when finishing running, I assume I would not get to get so many CIP in the gel (if have should be trace amount). Could you by any chance explain it to me? Thanks a lot
in the past I have found problems ligating gel purified fragments with dephos vectors. I have resolved this by treating the gel purified fragment with polynucleotide kinase (this can be done in ligase buffer) or preferably not performing a gel purification of the insert and just setting up the ligation with a 1:1 ratio (instead of 1:3) so that there is greater emphasis on the receptor vector than usual, then digesting the donor vector away after ligation (ie cut with something not in the gene or in receptor vector, but that will linearise any re-annealed donor vector.
Both of these methods have been succesful on a number of occassions for me.
good luck
jenjon
Thanks mate, you idea would be worth trying if I can find a suitable enzymes to cut, and alternatively need to make sure I have the polynucleotide kinase. A further question, here you still need to remove the phospho group on the receptor vector to allow ligation right? So which Alkaline Phosphotase did you use and from which company? Did you just add the AP into receptor vector and used the treated product for ligation directly? If you could give a protocol you referred to, that would be very help helpful, otherwise, I may come across some optimization process...which again take a while.... Thanks a lot
in the past I have found problems ligating gel purified fragments with dephos vectors. I have resolved this by treating the gel purified fragment with polynucleotide kinase (this can be done in ligase buffer) or preferably not performing a gel purification of the insert and just setting up the ligation with a 1:1 ratio (instead of 1:3) so that there is greater emphasis on the receptor vector than usual, then digesting the donor vector away after ligation (ie cut with something not in the gene or in receptor vector, but that will linearise any re-annealed donor vector.
Both of these methods have been succesful on a number of occassions for me.
good luck
jenjon
Thanks mate, you idea would be worth trying if I can find a suitable enzymes to cut, and alternatively need to make sure I have the polynucleotide kinase. A further question, here you still need to remove the phospho group on the receptor vector to allow ligation right? So which Alkaline Phosphotase did you use and from which company? Did you just add the AP into receptor vector and used the treated product for ligation directly? If you could give a protocol you referred to, that would be very help helpful, otherwise, I may come across some optimization process...which again take a while.... Thanks a lot
Hi
since you are doing a Bam/Bam insertion I would definately still dephos your receptor vector or it will probably just re-ligate itself. (I tend to use directional cloning to avoid this where possible) I use CIAP from NEB. I just throw approx 1ul into the digestion mix (after the RE digest has finished) - no need for buffer change and incubate for a further hour at 37C. I then heat inactivate at 65C for 30 min then clean up the DNA with a desalting kit (eg Qiagen nucleotide removal/PCR clean up kits).
If you want to polynucleotide kinase treat the gel purified insert – again I use this from NEB- just add the appropriate volume of ligase buffer (negates need to add extra ATP) to the sample (I use T4 ligase from promega), add the required amount of kinase (this will depend on the amount of DNA you have – the NEB datasheet is quite useful) and incubate 37C 30 minutes, then heat inactivate 65C 20 min. Since the sample is already in ligase buffer there is no need to clean up.
I think the theory is than if you gel purify a digested fragment it can be a bit rough on the overhanging ends. If you have knocked the phosphates off the vector than you are cloning into, you need to make sure that there are phosphates on the insert or ligation won’t happen. I have no idea if this is true, however I have found the technique to work….
jenjohn