Someone has just posted an Ultimate Molecular Cloning Guides online. Just google "Ultimate Molecular Cloning Guides" and you will find that. It seems useful for most molecular biologists.

Is it possible that if I have not included 6-8 bases near the RE site during primer designing, I can have trouble during ligation?
if the RE is not able to cut properly , then my insert and vector wont ligate well?
I am having trouble with my ligation.
I use 1:3 molar vector:insert ratio, 50 ng of vector in the system.
I also dephosphorylate the plasmid..
I have tried overnight ligation at 18degree C and rapid ligation at 22 degree C for an hour
if i transform only the plasmid i get colonies..
i also purify the pcr products
so my cells are OK
the only problem i see is that the ligation is not happening
is it faullty primer design??
am i stressing out too much and is it something simpler which is causing the problem ?? :sad_cry:

any suggestions??

Please can I get an idea/strategy on how to identify and select a plasmid that would carry a particular gene of interest into diverse microbial population simultaneously?

Ok folks, this is something I'm a bit unclear on.

I've been using CODEhop for designing my degenerate primers to clone a gene out of alligator.

Problem... I picked a gene that's small (~500bp) so there's not as many occurrences of conserved regions desirable enough to make more than one primer. At least that's what CODEhop tells me.

I've added the seq from Gallus, a few other aves, some mammals and xenopus for good measure of diversity.

Now I don't know the intricacies of codehop, but i'm assuming it takes into account potential hairpin loops or complementarity among the primer pair. Perhaps more so the former.

With the sequences in Clustal, I see multiple conserved regions, which vary from 4-5 amino acids with an occasional single aa, yet codehop won't capitalize on them.

I tried to copy and paste the conserved fragments and got some primers but are these usable? Any suggestions?

Hi,

I am looking to conduct an experiment involving satellite colonies. The experiment is based off the pGLO transformation lab that inserts an ampicillin resistance gene, araC, and GFP gene into E. coli. Once grown in LB/amp/ara culture, the transformed E. coli secrete beta lactamase, which breaks down the surrounding ampicillin and allows non-transformed "satellite" bacteria to grow around it.

I'm having some trouble coming up with a suitable experiment to research the satellites. So far my ideas are to investigate the correlation between the ampicillin concentration and number/amount of satellite colonies. So if the ampicillin is increased, the number of satellites should go down. But how would I measure the satellites? Should I count them, measure the radius of the whole colony, or test for the presence of beta lactamase?

If you have any ideas, please help! Thanks!