Cloning & Expression - Cloning & Expression (Mar/06/2007 )

hello all,
i am about to start the cloning and expression part of my work and have some doubts about it and would request any help or suggestions i can get.

1) i have to amplify 2.3kbp of fragment into a vector and then get it sequenced, i was planning to use deep vent taq for the process and then as deep vent taq make blunt ends, clone it into topo vector and send t for sequencing.

i would like to know if the deep vent is good enough for such a large amplification or should i choose some other enzyme, the amplified product should have as less apossible mutations, as possible and the gc content of the fragment amplified is :
Length = 2300 base pairs
Molecular Weight = 697469.00 Daltons, single stranded
Molecular Weight = 1396948.00 Daltons, double stranded
G+C content = 46.78%
A+T content = 53.22%

Nucleotide Number Mol%
A 543 23.61
C 556 24.17
G 520 22.61
T 681 29.61
so the question is the taq taht i have chossen good and also the vector.... the things are less mutations in the product and also easily cloned into topo.


thekid

well, 2.3 kb is a fairly long PCR but give it a go - if you have designed your primers well, there's no reason why it shouldn't work. Since you are planning on TOPO cloning it, you could try using a mixed enzyme like Expand, which has high fidelity and leaves A overhangs, or alternatively just add the A overhangs afterwards, it seems to work pretty well. Whether you get any mutations may also depend on the insert, if E. coli don't particularly like the insert then you may get some mutations. Once again, the only way to be sure it to try it.
I am not sure about expression, but as far as ligation goes, TOPO is a pretty foolproof system.

Sounds okay. But everything depends on the primer design. Bad design and woe becomes you.

And by the way, please don't call deep vent, taq. Deep vent isn't taq... Taq comes from Thermophilus aquaticus and deep vent is the commercial name for a polymerase derived from a Pyrococcus species.

You can not use taq for gene cloning work, unless it is mixed with a proof reading polymerase, or a protein fusion.

helllo,
thank you perneseblue for the info about deep vent enzyme and also scrat for the input about the problems and the things to be kept in mind as i am using primers that have already been published. i think i have less fear about the primers, even though i agree with scrat that primers are the most building blocks in cloning.

however i would like to know which enzyme i can use for the amplification of such a long product....
any help in this matter would be nice, i checked on expand as suggested by scrat.. but i would also like to know if there are any other enzymes that i can check in to.

thekid

Design primers carefully, and you should be fine with the PCR and other cloning steps.

Good Luck !!!

Phusion polymerase by Finnzymes is brilliant for strong amplification and excellent fidelity. I've isolated several products over 2 kb with this enzyme and it will not stuff up on you. If you are worried about size, design internal primers to amplify the product in 2 fragments and join them together via a 3-way ligation or splice overlap PCR.

hello,
ok, thanks you for all that suggestions... u know working on all the details for the cloning and the expression has been hectic and then i relaised that i have never found any help on how to go about designing a primer for expression, i mean how amny book s tell u that u have to keep a close eye on the base pair or else there wil be a shift and to check both the goi and vector for restriciton enzyme sites before proceding, keep this many number for primer ends so re is able to cut and all that......

i would really like to know if anybody has ever read in detail how you go ahead designing a primers for expression into any vector... i have never found any i learnt it from a friend and now i am doing it and so am making up a sop for it so others can use it. if anybody has read anything about such a designing guidelines, please let me know..


thekid