Is a 3.5 kb fragemnt too big to be cloned ? - (Jul/16/2011 )

I'm trying to clone a 3.5 fragment (resulted of a double digest by BamHI and KpnI) into another vector which is 7.5 kb big. all I'v got so far has been vector self religation. I'm wondering if it's generally troublesome to clone a fragment as big as this or I still need to optimize my procedure !
it's worth mentioning that I do dephosphorylation of my vector by fastAP from Fermentase before ligation. Also the insert:vector molar ratio in my ligation reaction is 3:1. Any ideas/suggestions?

Thanks
Opal

3.5 kb should be no problem in general.
Are you sure you efficiently inactivated the Phosphatase? ...fastAP? ...sounds like Antarctic Phosphatas? ...normally inactivation should be no problem.

You checked if vector and insert are properly digested by self-ligation and checking that on an agarose gel?

Regards,
p

Fast AP is FastAP™ Thermosensitive Alkaline Phosphatase; according to the manual it inactivates by 20min at 65'C. The insert can not cause the problem since its original vector has a different antibiotic resistance. for the vector I do sequential digestion first with BamHI 3 hours 60 U for 3 mg of vector (usually 10 is recommended for 1 mg); then column purified and overnight digested with half of this amount of Kpn1 the day after again I add some kpn1 for 1 or 2 hours! so it should be completely digested but apparently a small proportion is still single digested or not digested. The question is how can I make the rest to end up with a correct ligation. Should I increase the insert/vector ratio above 3 times?

It is very difficult to completely cut a vector. Since uncut vector transforms with the same resistance as your desired product, this makes analysis of ligation products tedious. You can assist this by making the initial vector easy to distinguish -- by adding an insert for expressing gfp, for example.

Instead of cutting a vector, it is better in most cases to amplify it with PCR primers. You can amplify the vector with any restriction sites you want (don't forget to add 4-6 bp of 5' sequence following the restriction site). You need only a very small amount of the original vector as a template. After the PCR, you need to purify the DNA and then cut with your two enzymes plus DpnI. The two enzymes prepare the ends of the PCR product, and the DpnI cuts the template DNA (but not your PCR product). If you choose enzymes that can be heat killed, this restriction product can be directly used in ligation. Otherwise, you need to purify the restriction enzyme digestion prior to ligation. This preparation need only be done once per target vector, and can be stored and used for many months.

for a vector which is 7.5 kb do you think this method will work? Also I've always had difficulty with cloning a pcr product therefore I always clone the fragment in a TA vector first and then from there I cut it and subclone it into the destination vector.

7.5 kb should be no problem for a Polymerase like e.g. Phusion.
To my point of view PCR amplification of the backbone is a good idea ...but also has its drawbacks (mutations introduced by PCR, ...) ...i would try to digest the vector and gel purify it you can also think about adding a restriction enzyme that specifically cuts your uncut vector (e.g. restriction sites that lie within the region of the MCS that you remove by digestion) into the ligation reaction (rarely did that ...but some people do that).

Background does occur in most cases ...but it's no problem most of the times when you have a primers for screening colonies (one primer on the backbone, the other primer binds to the insert) ...using PCR you can screen many colonies at once and easily find positive clones.

If you don't find any positive colonies and you can rule out that the insert and vector are properly cut (do self ligate) than you have to consider if your insert is toxic under the conditions used.

Regards,
p

Mutations in the vector are usually don't cares. Either they are lethal, and there is no transformation, or they are inconsequential, since you aren't planning on using the vector again in any case. Rarely would a viable mutation in a vector (as opposed to the insert) be a problem.

it always depends on what you are cloning in which backbone ...but most of the time the insert isn't totally isolated from the rest of the backbone (promoter, terminator, palindormic protein binding sites, etc.) ...so you'll have to care about mutations and keep in mind that PCR could have introduced such a mutation (that then has been selected for) when trouble shooting.

But i agree ...usually it is not a problem ...always depends on how nasty the insert is.

Regards,
p

I got my clone! Thanks every body for your help! I just had to screen many colonies by direct PCR and finally it was there !!!