help with moving an insert to a new vector - (Apr/21/2007 )

I want to move an insert (3.2 kb) from one vector (pUC-19) to another (pET-Duet ~6 kb). My pET-Duet vector already has an insert where I want to put the new one so I thought double digestion of both insert1/pUC-19 and insert2/pET-Duet would be a convenient way to move the gene and not have to worry about the recipient vector not being completely digested.

What I did:

1. double digest both a miniprep of both plasmids with Invitrogen NcoI and BamHI for 3 hours
2. gel purify both digests, cutting out the liberated vector and insert
3. dephosphorylate pET-Duet with Promega CIAP then PCR Cleanup
4. phosphorylate insert, just to be sure, with Invitrogen T4 kinase
5. measure insert and vector concentrations on a gel
6. set up 20 uL ligations using 0.2 uL NEB ligase O/N at 16 C 3:1 and 4:1 insert:vector
7. desalt ligations by precipitation and resuspend pellets in 10 uL ddH2O
8. check ligation by running 5 uL on a gel
9. electroporate 1 uL into BL21(DE3) 2500V in 0.2cm Biorad cuvette time constant ~5.4 - 5.8 with 30 minute rescue in SOC at 37 C and 250 rpm
10. plate O/N at 37 C on LB/Amp

Results:

1. Diluted supercoiled pUC-19 gave ~200 colonies for 50 uL of rescue broth
2. double digested vector, no ligase gave no colonies
3. double digested vector religation check gave no colonies
4 insert no ligase gave no colonies
5. insert religation check gave no colonies
6. vector + insert gave no colonies

The ligation gel shows that all reactions with ligase caused DNA to appear larger than the non-ligated vector and insert. Each lane showed multiple bands. Restriction enzymes seems ok because I get the expected digest products. Ligase seems ok because treated DNA migrates slower on the gel. Cells and plates seem ok because uncut pUC transforms E. coli. I have done this several times with the same result. I have done similar procedures before for this type of experiment as well as cloning of PCR fragments by both blunt and sticky end ligation. Can someone tell me where the problem may be?

brewer

I feel for you, it looks like you've got a tightly controlled experiment but things aren't working out. I don't know how much help I (or anyone else) can be in this circumstance but I do have a few comments:

1. What cells are you transforming into? Are you transforming into a cloning strain (ex DH5a) or an expression strain (BL21)? The reason I ask this is that it appears that your ligation/transformation works but the cells aren't surviving post-transformation. Could it be a lethal insert (the T7lac promoter of pETDUET is muh stronger than the pUC lac promoter). In most strains, and particularly in expression strains, addition of glucose will repress expression of the insert and (in BL21) the T7 polymerase.

2. Gel purification: are you sure you're not destabilizing the DNA by exposing it to short wave UV (312 or 302 nm)? Double-check that your lamp/transluminator is set to 365 nm or use a non-UV dye (Methylene blue) to stain your DNA.

3. You've got a lot of negative controls but no positive controls for: (1) the ligation (2) transformation of pETDUET

i) maybe cut pUC with SmaI and HincII (both blunt), gel purify. Then, religate with the controls below. If you get false positives, put the enzymes in the ligation mix (see Sambrook for the protocol)... but you probably won't.

ii) You did the pUC transformation control (good) but pETDUET is bigger and might be lower efficiency. You mentioned it was diluted pUC, but how dilute? Calculate the number of CFU/ug. If it's less than 10E6 that might be the problem.

4. I only mention this because it once happened to me: double check your plates that you are plating on Amp plates and not some other antibiotic.

Good luck

Hi,

I'd suggest simplifying a few of the steps you've taken. I'd skip step 3, 4 and 5. These steps tend to waste DNAs for me.

Since you're using two different REs, you don't really have to worry about self-ligation. Dephosphorylation of insert is very minimal when you make it fresh, within a day or two or three. So, phosphorylation is not necessary.

Measuring the conc. and working out the ratio could be practised but I tend to just add 100 to 150 ng of vector and then the whole of insert DNA into the ligation rxn. Always worked for me.

Bests