No insert for cloning PCR product into expression vector - (Nov/08/2005 )

I have been trying to put 2 inserts into some GFP vectors for making 2 fusion proteins without success for quite some time. Here is my protocol:
1. PCR amplify my 3.5 kb or 450bp inserts with flanking RE sites on the primers. First I tried EcoRV and BamHI after few tries with no success I ordered new primers and used SacII and BamHI still no luck. I included 5 extra bases on the other side of the restriction site to ensure cutting usually I use 5 A's or 5 T's in a row.
2. Run an aliquot on gel to make sure band size is as expected. And they are with good yield. Used to have a PCR clean step here but if I do that, I always lose so much DNA that after digestion and gel purification, not enough insert is left so I skipped it hoping it would still make the digestion OK. I am not sure if that's true???
3. Digest the rest of the 50uL from PCR with both enzymes at the same time for at least 3 hrs (I also tried first SacII in BamHI buffer overnight which should be 100% activity according to NEB catalog then 3 hrs of BamHI the next day). I also digest the vectors at the same time.
4. Gel purify vectors and insert, check concentration after purification
5. Setup ligation at insert to vector ratio of 3:1 at 16 degrees O/N in 20uL.
6. Transform 10uL into 100uL XL-1 competent cells and plate on appropriate plates.

I always get more colonies on my vector plus ligase plate than I'd like (at least 20 sometimes up to 70) and I do get like at least 3 times as many colonies on my insert plates but they are always negative for the 3.5kb insert and I've gotten the 450bp insert to incorporate I believe.
Can anyone suggest anything? I am so desperate. I just don't know what is wrong.

vivian - do you phoshpatase your vector?

and also, I find that gel-purifying your pcr should work OK for ligating. I have not cloned much recently, but I used to do it allllll the time, and I never used a PCR clean-up, I just pulled the fragment from the gel and went to the next step.

what about running a gob of your PCR product, gel-purifying the band, then proceeding to the digestion? I think perhaps you are seeing inhibition from PCR reaction components, for your RE step? I don't know if this will help, but that is the way I would do it.

good luck

Hi
Don´t get desperate, we will solve the problem.
First thing I can say is that it looks you have a non digested or partially digested plasmid, reason why you have so many colonies when transforming bacteria only with it without insert.

I don´t have here the biolabs catalog with me but tomorrow I will look it up the characteristics of those enzymes. If I had I could check for you.
From which company are the enzymes. Check that and see the buffer recommended for each of the enzyme you are using.
VERY IMPORTANT is to check the type of buffer each enzyme need. The bigger difference between different buffer is the amount of salt; it can vary from 50mM to 150mM NaCl.
In case sac II and Bam HI require different amounts of salt, then you must do a sequencial digestion (see biolabs catalog for information on this), starting with the enzyme that needs less salt.

I mention the name of the company because some enzymes vary slightly acording to the provider, at least in my hands.

I can tell you that BamHI and EcoRV for me work perfect when using buffer React 3 from Invitrogen.
I don´t know why you had problems with those.

With sacII never worked before but I tell something you should do.
You sould know which of the 2 enzymes is not cuting properly.
In order to know that digest your plasmid either with BamHI, or sacII or both enzymes, run all digeted products and next to them run also non-digested vector.

I will tell you what I do:

- do PCR with primers containing restriction sites (25 ul or 50 ul final volume) using proof reading enzyme (very important!!).
- run 5% on gel to check size
- usually I freeze the PCR sample (-20C) until I am ready to start a long day of digesting DNA, purifying from gel, quantification and ligation.
-digest plasmid and insert.
(plasmid digest not only double but aso single digestion in case one of the enzymes was not working you know immeditly and don´t continue further).
- If didn´t digest can be because not appropriate buffer was used, not enough enzyme, enzyme has expired or is not working because was not properly stored or enzyme is sensitive to methylation.

- I usually digest only 1ug plasmid DNA with 10 units each enzyme (usually 1 ul) in a final volume of 50ul and incubate for 2 hours
( Yes i use a lot of DNA, I do this only for cloning porposes, when is only to check plasmids whether they have insert or not, I use only 1 unit enzyme and is always enough!

- after digestion I run on 0.7 % gel everything (50ul) using large slots.

- run the gel quite long to make sure I can cut the right band.

- after gel run enough put gel on top of UV light but with glass plate underneath the gel because some UV lamps are very strong and therefore you can damage the DNA and have problems in cloning afterwards. (note: glass t«doesn´t allow to see DNA bands so clearly but you can still see)

- cut the band as quick as possible and as thin as possible

- and only after cuting gel and puting it inside a tube I take a picture of the rest of the gel. Then you can see if you cut right and check single digestion plasmids...

- meanwhile while the DNA plasmid is runing I purify the Digested PCR sample with column, there are columns suitable for this. therefore you get rid of all the tiny litle parts of nucleotide sequence unwanted that can easily religate again.
- this way you don´t loose a lot of DNA.

- isolate and purify DNA from gel elute in 40 or 50ul H2O

- run 2ul each DNA in gel along with mass ladder to estimate DNA concentration

- prepare ligatio reaction (50ng vector ; insert:DNA ratio=3 or 5)
- ligation ON 16C

- day after transform 100ul competent cells with all ligation reaction (10ul)
(Sambrook usually recommends for normal transformation not exceeding 25 ng per 50 ul competent cells neither exceeding 5% of cells volume; I know here I am using 10% DNA but I just do this with DNA from ligation not for intact plasmids)

- plate all transformants in plate (1 ml) with right antibiotic

VERY IMPORTANT: check whether sacII is methylation sensitive, last 2 weeks I was trying to clone a gene using BamHI and XbaI and I could never digest with XbaI. Now I know why!!!

Is it the only time you are having problems cloning or you had problems with others inserts also.

Good luck, let me know if it works,

Hi again,

Just remembered to say that is better to plate intead of all transformants in a plate, plate 10% or 20% in ne plate and the rest in another. I just plate everything when I treat digested vector with alkaline phosphatase.
Sorry,

Good luck

I thought I'd stay away from digesting my PCR product because I may have incomplete digestion on my PCR product or on the vectors hence the high vector+ligase background. (Although I always do parallel single digests to ensure each enzyme is cutting properly and they each appear to linearize my vector just fine) So I ordered the zero blunt TOPO kit based on topoisomerase from Invitrogen hoping is would solve my problem. It worked great for my smaller insert clone (450bp) 6/6 were positive but once again, the large clone (3kb) is all negative for insert (0/16 in two transformation from 1 PCR rxn). The kit uses TOP10 cells and I was using XL-1 cells before. I switched to EcoRV and BamHI, gel after PCR looks clean with only one fairly bright band at expected size and a much lower band at <100bp presuming primer dimers? I've never seen this small band before out of the 10 times i've done this PCR but maybe I just missed it for whatever reason. Still, even with this smaller band, I should still see at least some clones with my 3kb insert right? I can't figure out why this insert is being so difficult. Different cells, different vectors, not using RE at all, I have no idea how to troubleshoot now. Has anyone had experience using this kit and can anyone give me any ideas?