Why my insert and vector dissappear after mini prep? - (Aug/06/2005 )
I am making a clones. The insert is 1.6kb and the vector is 6.5Kb.After ligation , I transform the plasmid into DH5a. The ratio of the colonies on the control plate and exp plate is aboout 3:2. So I pick up 10 colony to do mini prep. After that, I run the mini prep extractions on the gel. It is strange that the bands showed up from all the colonies is about 1.5Kb. Obiously it is not the insert or the vector. Then I digest the mini prep extractions and run gel again. The bands showed up again are all the same as before.
Does anyone give me any suggestion where is my insert and vector???
Thanks a lot!
Sorry to bother.
I think my case again and make sure that the bands are not the inserts. Now I suspect that the bands showed up are probably the supercoiled vector. However I don't know whether the supercoiled vector (6.5kb)can show 1.5kb on the gel.
Does anyone give any idea on my case??
Thanks a lot!
It could be the supercoiled vector on the gel - they can run at a variety of sizes.
Can you check your plasmid preps by PCR for your insert? Or even screen the colonies by PCR before picking them for plasmid prep? Digestion takes up alot of your plasmid, and I find PCR to be just as good for checking sizes of inserts (in fact, I never do digestion for inserts!).
Colony PCR is an easy way of checking many clones, as long as you have PCR primers to work with. You just set up a standard PCR mix, making sure that the MgCl2 is between 1.5mM and 2mM. Aliquot this into PCR tubes (on ice). At the same time, have a new agar plate with numbers labelled for the number of colonies you want to test. (I have done up to 2 x 96 plates at a time). Using a sterile toothpick (I use the pointy ones) transfer the colony from your plate, into the PCR mix, twirl, then streak onto your plate in the appropriately numbered spot. You don't need much bacteria for either the PCR or the new plate, so a streak of 0.5-1cm is enough. You can then run your PCR and check the sizes of inserts. If you set this up early enough in the day, you will know which colonies have the inserts of interest, and just before you leave you can pick a colony from the new plate into an overnight culture for plasmid prep.
For a "Standard" reaction I use T7 and SP6 primers (will work with all my clones, regardless of insert) and cresol red in my PCR (so I don't have to add loading buffer) and do PCR (95 C 5 min; (95 C 30sec, 55 C 30sec, 72 C 45sec-1min)x30; 72 C 5min). I have used this to screen almost 400 clones a day. The hardest bit was not getting mixed up on the replicator plates! For that I recommend colour markers and working in the fume-hood so no-one disturbs you
Some vectors such as Lentiviral with strong LTRs give problem with non Rec- cells. Try using XL1Blue or Stbl2 for your ligation mix. These cells are few recombinase negative cells which avoid susceptibility of cells recombining due to vectors..
Hi, Nic T,
That is really great idea for me! Thank you very much for good advice!