Designing forward primer incorporating Nde1 cut site - Tips on cloning with Nde1? (Feb/11/2007 )

Hi

I'm trying to clone a 900 bp fragment into the Novagen pEt15b vector. We have the vector with a another gene cloned into it between Nde1 and BamH1 sites, so i'm going to have to digest it out, and then put my insert (from PCR) back into it. from what i've seen in the literature and in this forum, cutting with Nde1 is a tough business and i have a few questions...

1. in designing a primer incorporating an Nde1 cut site, could i use the start codon that is native to the Nde1 cut site (CATATG) and insert a start codon for the gene immediately after the cut site? or do i just use the Nde1 "start" codon? or perhaps insert a Kozak sequence between the cut site and a second start codon (keeping the start codon in frame)?

5'-CAACTCGAG CATATG ATG (+/-) CCG AAG .......3' ???


2. i've seen that NEB says a double digest is okay with Bam and Nde1 (and BSA) but would i be better off doing a sequential with a phenol-chloroform gel purification step in between?

3. it seems like Nde1 needs at least seven bases upstream of its cut site for efficient digestion. i've incorporated nine bases upstream of the cut site, but in reading the literature i've seen that most groups subclone the PCR insert into an A/T vector such as TOPO TA and then cut that insert out and ligate into the pET vector. i guess my question is should i still do the subcloning step or, because i've incorporated the extra bases upstream of the cut site, go straight to the pET ligation? and if i were to do the subcloning step, would the TOPO TA vector be a good choice?

i'm relatively new to cloning practices (being a chemist ) and i'm trying to think this one wayyyy through before i embark on what seems like a journey through cloning hell. any help anyone could give me wil be much appreciated and may the cloning fairy smile on all your endeavors

---

Yes. you can use the ATG native to the NdeI site as the start codon. NdeI site is often used in similar way.
I am uncertain how much leeway your protein has, and so unable answer the second part of this question, regarding the Kozak sequence.

Concensus Kozak sequence
gccgcc(A/G)ccAUGG

However, I am guessing that your gene will go into an expression vector. In such a situation the promoter may be strong enough (somekind of overpowering super engineered viral promoter and enhancer) to make kozak sequence dispensible. (But please do check, it might not be so)



I would do a double digest. The buffer chosen would be NdeI's favoured buffer. Conduct you digest in a suitably large volume. Overly concentrated DNA doesn't cut well. Don't use too much enzyme (BamHI especially), watch the total glycerol concentration (which should not exceed 5%). And give NdeI enough time to cut. It is far slower then BamHI, EcoRI, XhoI or similar enzymes. I would do the double digest overnight and be done with it.



This is a personal choice but I would clone the fragment directly. TA cloning in an extra step, which I can ill afford. Most people do not take NdeI extra requirements into considerations, and thus run afoul in their cloning. Similar situation occurs with Not, which also requires longer nt buffers around the restriction site. However as you have already taken into consideration of NdeI's requirements, I would say you should not have any cutting problem. However do run a check on your primers to make sure that there are no self annealing. Oligo calculator

It'll make PCR alot easier. (fewer secondary bands)

The more steps involved in cloning, the more likely for things to go wrong.




Well good luck.

PS. And the deity of molecular biology aren't fairies. They are GODS!!!
Be warned! Calling them fairies or lesser beings might bring down their wrath on you . And believe me you don't want that.
Invoke any magic protection you have (eg magic underwear, magic amulet, holy icon or idol) The gods accept sacrifices of blood, sweat and tears (preferably not your own), coffee, kit-kat bars, coke, and human lifespan/lifeforce.

---

Thanks much for the info. i think i'll use the Nde1 native ATG and be done with it since i actually have the primers all set to go-- no self annealing . as for doing the subcloning step, i usually get tons of the correct PCR product as i use a plasmid template and i might just do it both ways with and without the subcloning step. (we also have relatively new TOPO TA vector sitting in the freezer and my grinch of an advisor will be appeased that i'm using it-- this whole cloning business is my personal venture, he wants me to use some other horrifically fickle vector for protein expression). i think i'll stick with an overnight double digest and run a gel to see how it goes.

as for personal sacrifices to the cloning deities, i have an unsuspecting undergrad whose science-untainted blood will do just fine.

---