I am trying to clone a 5.1 kb insert into a 13.5 kb vector ( plant binary vector) using SacII site. My recipient vector is a plant binary vector (pPIPRA560) and my experimental procedures were as follows:
1. I digested 5 µl of miniprep DNA (concentration around 250-300 ng/µl) in a 20 µl reaction using 2 µl of SacII for 4 hrs. Heat denature the restriction enzyme at 65 oC for 20 minutes and add 1 µl of TSAP (Promega) and incubated at 37 oC for dephosphorylaton. After dephosphorylation, I incubated the total reaction mixture at 74 oC for 15 minutes to inactivate the TSAP.
2. For isolating insert I digested 15 µl of miniprep DNA using 3 µl of SacII for 4 hrs and then incubated at 65 oC to inactivate the restriction enzyme. After heat inactivation of restriction enzyme I run reaction mixture in 1% TAE gel and isolated my desired band by gel extraction and purification using Promega SV gel and PCR purification kit.
3. I ran my prepared vector and insert in 1% agarose gel to check the size again and in gel they look okay
4. After confirming my right size of the vector and insert, I set up ligation using various ration like 1:3, 1:4, 1:5, 1:6 etc.
5. I transform 5 µl of ligation mixture into 50 µl of competent E coli cells.
6. Screened lots of colonies (150-200) but none was positive.
7. I used standard ligase from Promega, concentrated ligase from NEB. I also used home made cell as well as commercially available competent cells.
8. Initially I used heat shock method and then I used electroporation using ElectroSHOX competent cells from Bioline.
9. I set up ligation at 16 oC, 4oC for overnight, for weekend.
10. I normally used 1 µl of ligase.
11. I checked my ligase, buffer and all are good.
12. One important concern is the Bt genes which are present in my recipient vector is toxic gene, one of them contain intron but the other not.
13. I am trying to do this clone for the last 6 months but still not successful while at the same time I have generated around 80 other constructs.
Can anyone please suggest to me how can I win this frustrating war?
Hi,
Try gel-purifying both plasmid and insert after digest/dephosphorylation, before ligation. And make sure your plasmid is linear (if you see several bands, only excise the linear DNA, which is usually the less diffuse band running slower than supercoiled, and faster than nicked).
If you are unsure what to look for, then run with uncut plasmid and plasmid cut with another good single cutting enzyme in two other lanes for comparison (and with a nicking enzyme in a third lane, if needed).
According to NEB (https://www.neb.com/products/r0157-sacii) SacII needs to interact with two copies of it's recognition sequence to be able to cut efficiently.
And just to be the devils advocate, you say you checked ligase, buffers and they were good. But if you get a lot of plasmid without the insert popping up, it could indicate that you either do not digest the plasmid (sufficiently), or that the plasmid can re-ligate. I don't have practical experience with this phosphatase (I've usually used alkaline- or antarctic phosphatase), but did you check the TSAP activity?
Hope this helps...
Cheers,
W
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology