TaylorDNA36

Hi Koeng,

Making new plasmids from scratch is not always an easy task, and there are lots of different elements to consider beforehand. But as some of the other posts have mentioned, figuring out why you are making it is key to making a good vector. If you take a look at biobricks (http://biobricks.org/) or oxford genetics (http://www.oxfordgenetics.co.uk/) you can see how the plasmids are designed to do XY and Z before the vectors were engineered. Check out the drop down box at the latter site and you can see what the guys who made it were trying to incorporate, but they must have sat down and decided this at the start! Good luck with the vector, hope its works out.  

Regards,

T

Introns and exons can exist within the promoter. Exons are simply the mRNA, not just the protein coding sequence. So it somply means that some of the mRNA is produced within the promtoer region, but necessarily the coding sequence of hte mRNA.

Eukaryotes promoters are quite complex and some binding sites for proteins that activate the transcription may actually be downstream of the mRNA start position, or it may be that the presensce of the first intron/exon increases the promoters activity and is therefore considered part of the promoter region. Many of the strongest mammalian promoters have introns in them, Ubiquitin, Elongation factor alpha and chicken beta actin (full length) all have introns in them. In fact, many people add an intron upstream of their gene to prolong expression in vivo. It makes a gene more like a natural gene and decreases its chances of being methylated.

I think the amount of DNA you are using is probably too low. Because the plasmid is at least 5 times bigger than those used in normal cloning 3-4kb you will need to add 5 times more DNA to get the same number of physical copies in your reaction. The cloning will also be much less eficient because the bacteria normally preferentially take up smaller DNA fragments. Your vector is pretty big.

I would be digesting at least 5ug of DNA for the reaction and add a lot more into the ligation and transformation. Also try the ligation overnight at 16 degrees, that might help but only a bit. I think doing the uncut DNA control would also be good to check the selection, plates electroporation etc.  Good luck!

It sounds like a failure in one of your reagents to me. The ligase buffer has ATP in that can degrade if you freeze thaw it too much, and you said it was quite old. Also, what was numerous colonies on the positive control? If you just took some pure plasmid DNA and transformed it as your positive control (but didnt get LOADS of colonies) then it may be that your cells are not that great. If they are not very competent anymore then the only stuff that will get in is the supercoiled DNA, hence you get very low background in your ligation (uncut from your digest), but some (not loads) of colonies from the positive control (where you added a lot of uncut DNA), but your ligated DNA (which will not be supercoiled) can struggle to get into the cells if they are poorly competent. I would calculate the efficiency of the transformation and see how good your cells are.
If you are new to cloning then there are some useful things at http://www.howtoclonedna.co.uk/, this might have some insights. The ligation protocol seems ok and gives some background to the controls. Dont think it tells you how to calculate the efficiency though. But I definitely think it sounds like low efficiency cloning to me based on your last post but knowing what the numerous colonies meant would maybe help.

I would try:
Changing ligase buffer
Using someone elses ligase that is known to work
Check you have DNA after digestion and clean up on a gel or by spec (but it will be low concentration)
Set up a few different ratios for the ligation (more fragment etc)
Use different clean up kits, sometimes the colunms can fail or the buffers go off

The problem is that if the ligation works you wont know which one it was that is broken! Hope this helps.

You could perform a PCR using a polydT primer and a primer that is specific for a region inside your virus genome. I would aim for a final product that is about 200 bp long (i.e. the forward primer binds 200 bp from the end of the virus genome), then just use a generic RT-PCR kit and run the product on a gel. This would not be quantitative but would tell you if you have any polyA there. You could also do it by QPCR but the probe would cost a bit. A separate set of QPCR primers and probe could also be run alongside the polyA set that binds to a known region within the virus genome. The ratio between this, and the one that binds the polyA, would give you a rough idea of the percentage that has a polyA compared to the total amount of virus genomes in the tube.