Ligation into EGFP problems - Ligation into EGFP problems (Jun/02/2004 )

I am having huge problems ligating a 0.9 kb XhoI/PstI fragment into an pIRES2-EGFP vector. I have succeded previously with no seeming problems, but now I want to replace that with a fragment with internal mutations. The problem seems to exist in that uncut EGFP migrates at the same rate as my double digested EGFP clone (which has released the previous 0.9 kb insert), so that everytime I gel purify, ligate and transform into XL10 Gold my vector vector control plate contains many colonies.

I have tried double digesting the original EGFP vector, which migrates the same as single cut EGFP (the XhoI and PstI sites are very close in the MCS) but different than uncut. I have gel purified this, followed with dephosphorylation of vector with SAP, yet still after ligation and transformation I am getting hundreds of vector-vector controls.

I have tried heating my DNA before digestion and ligation, triple digests, differing % agarose, running digests longer, and cutting a very small fraction out of the agarose, yet still I have the same problem. Any ideas someone ?

Many Thanks in Advance,
CrazyKiwiDave

Tell me your Vector information (preferably catalog No.) and the Insert information, and which Restriction Enzyme Sites in the Vector MCS are also in your Insert Sequence, I will try to figure out for you.

The vector is pIRES2-EGFP cat#6029-1, and the insert is in pocus, and when cut with XhoI/PstI yields a 900 bp fragment. Previously I used XhoI/PstI cut pIRES2-EGFP to ligate the fragment, and it worked first time. This time I try both pIRES2-EGFP and the vector I had previously constructed cut with XhoI/PstI. I have checked the transformant colonies and they contain the vector without insert.

Cheers
CrazyKiwiDave

NB: Clonetech (now BD vector#6029-1)

CrazyKiwiDave,

Before the work, please make sure you have a good reagents.

You could do duble digestion or digest sequentially.

For double digestion, you need to check where the enzymes from. If it was from New England Biolabs, you can use the buffer they suggested. If it still does not work, or the enzymes were from other vendors, may be sequential digestion is the best and safest way to do, even it cost you a little more time.

For sequential digestion, there are a lot of steps, each steps will incur some vector or insert loss, therefore, try to begin with large amount of initial plasmid and inserts (just remind you, if your insert is in other vector, it will be very easy to verify your target insert before ligation; if it is from PCR product, be sure you have the suitable clamp nucleotides, and adjust the digestion accordingly).

Belows are protocol based upon the vector Clontech pIRES2-EGFP and Insert from PCR product, which I think is a little more tricky than fron other vector.

1. Get purified plasmid. Phenol and cloroform extraction, followed by ethanol precipitation of PCR product;

2. In 100 microlitter, digestion of 50 microgram of pIRES2-EGFP and 10 microgram PCR products (roughly Five 50 microlitter middle to high level PCR reactions) with 10 microlitter of one enzyme overnight;

3. Run the digested products against the marker and the pre-digested products, especially plasmid, to confirm the completion of digestion;

4. Phenol and cloroform extraction, followed by ethanol precipitation of digested plasmid and PCR product. Dissolve the pellet in the ultra-pure water. Also, you may alliquot a little for gel running on step 6, depend on you case. Right now, you have about 40 microgram of digested pIRES2-EGFP and 8 microgram digested PCR products. ;

5. In 100 microlitter, digestion of 40 microgram of digested pIRES2-EGFP and 8 microgram digested PCR products with 10 microlitter of the other enzyme overnight;

6. If you have a very tiny fragment released, you do NOT need to run the gel; If you have a obvious size of fragment, run the double digested vector and PCR products against the marker and the single digested products;

7. Gel purification of double digested plasmid and PCR product;

8. Check the yields of double digested plasmid and PCR product, and determinate their concentrations. You will have about 30 microgram of digested pIRES2-EGFP and 6 microgram digested PCR products;

9. Go ahead with ligation with about 3:1 (prefered) to 1:3 ratio of insert : vector MOLAR concentions. Also very important, you must include double digested vector only (without ligase and insert) control and no insert (with ligase and double digested vector) control. Transformation the above three ligation reaction into the competent cells, respectively.

The above guideline is very concervative, but usually very effective. To begin with lager amount of DNA could give you much room for trouble shooting.

xho1 does not like to cut near the end of DNA fragments (very, very picky in my hands). If you use the empty vector cut with Xho1 (1h) first then with Pst1(1h more). You should beable to use the same buffer (Pst1 buffer is best to prevent star activity?). I don't use SAP but CIP (1.5u) which i just add to my digest for 40' 37C (maybe SAP will work like that also?). Gel purify only after all modifications with the next step the ligation (so it is nice and clean, prevent any impurities like ethanol or what ever u use for gel isolation). Ligate o/n at 16C. Keep gycerol conc. (from ligase) below 1/10 (max) (Prevent any ligase on the out side of your pipette tip ).

I usually cut 3ug in 150 ul total volume. This dilutes any impurities and virtually always cuts well (10X over digestion so 3ul enzyme). It also prevents the temptation to overload the gel (You'll need about a 1 inch wide, thick gel comb to load it, at a minimum). Overloaded gels (where you get streaky patterns instead of a nice sharp clean band) are, in my opinion,
pointless to separate uncut vector from the digest.