Purification and cloing of large fragments - cloning (Dec/08/2006 )
I was trying to clone a 10kb fragment into a 12kb vector, but had no luck. Any suggestions? One of the problems I had is that I can never get a good yield with the Qiagen gel extraction kit. I aslo tried QiaEXII, but it gave me similar yields. Also what ligation kit, competent cells do people usually use?
Many thanks!
I feel Qiagen kit is OK.
You might elute DNA with water or 1/10 elution buffer in 30 ul, if necessary, dry DNA then dissovled in less amount water for increasing DNA concentration before ligation
i would use Genehog in a situation like this. Also go for electroporation rather then chemical transformation. As the parent vector is significantly smaller then the vector+insert plasmid, any self ligated plasmid is more likely to transform. You need the better transformation efficiency of electroporation to over come this bias.
As for fragment recovery, a labmate of mine, simply takes the hit in recovery efficiency (as column purification is so convenient). He simply begins with far more DNA then needed, losses a bit but still ends up with enough DNA to work with.
If the above suggestion is not practical, how about eletrolution? You have virtually 100% recovery, though it takes much much longer then simple column purification.
Many thanks!
For large fragments (10-50 kb) I use QIAEXII with very good yields! I don’t know if you do it, but to increase yield, I heat the water or TE before eluting, and/or after adding it I incubate at 50°C for 3-5 min before centrifugation to collect my DNA. And I don’t vortex, I mix by hand.
Thy increasing the amount of DNA you are digesting.
By the way, when I started my Master studies, QIAEXII wasn’t working very well, but it was because the kit was a little bit old and the ethanol was evaporated in the buffer PE (wash buffer).