Update on work

So far....

I have been hard at working harvesting cut plasmid and insert using various methods. I combined these in a ligation reaction and transformed some E. coli with my mixture. Unfortunately the colonies I screened did not contain my insert, meaning the vector closed without the insert. This is weird because I cut my plasmid with EcoRI and BamHI which have different recognition sites which ideally means they shouldn't get ligated back together, however we do not live in a perfect work so I suspect my plasmid wasn't completely cut with both enzymes. This would cause two things, first it would mean that its nearly impossible for my insert to be ligated into the vector and second that my vector can be ligated back together. My problem is that my restriction cut sights are so close together (2 bp) that I cannot tell by gel electrophoresis whether both enzymes have truly cut the plasmid and I have read that cut sites this close can cause problems because the RE (restriction endonucleases) cannot digest both. So to combat this I have decided to use different RE that have 20 bp inbetween them and see how that works. This means I need to redigest both my vector and insert and do gel extractions... which I dread... So I'll be hard at work the next week lol. Sorry about the lack of posts, I didn't realize how busy I would be, however I will try to post more!!

Keep commenting on my posts as it motivates me to post more!!