I am trying to clone a 300 bp gene into pTYB2 vector (7.4kb)for later protein purification method by IMPACT-CN. NdeI (producing sticky end) and SmaI (blunt end) restriction sites were constructed into primers corresponding to the N- and C- termini of the gene and incorporated by PCR amplification. The PCR product and the vector were both double digested with SmaI and NdeI respectively. After gel purification, the digested insert (gene) and the vector were used to perform ligation with T4 DNA ligase. Since the expected product is not very different from the digested vector, it was really hard to tell whether the desired product was present on the gel. Then the ligation mixture was used to transform to JM109 competent cells. NO COLONIES WERE GROWING. :cry: I have done a bunch of controls. It seems that I have issue with either ligation or transformation, or both. I really hope someone here can help me with this. Molecular biology is all about experiences, great care and luck.
Here are the procedures.
(1). Double digestion
2 ug DNA
SmaI 25 degree C digestion for 2.5 hours
increase the temperature to 37 degree C, add NdeI, continue the digestion for 2.5 hours
deactivate the enzymes by heating at 65 degree C for 20 min
(2). Gel purification of digested products
phenol/chloroform extraction (to remove the enzyme)-->EtOH precipitation (with NaOAc added)-->spin down the DNA-->70% EtOH cold wash-->dry in SpeedVac-->resuspend in 20 uL of TE-->add agarose dye,load on 1% agarose gel-->cut the band under UV light-->electroelution using dialysis tubing-->EtOH precipitation70% EtOH cold wash-->dry in SpeedVac-->resuspend in 20 uL of TE
(3). Run a 1% agarose gel with known concentration sample to estimate the concentration of insert and vector
(4). Ligation with T4 DNA ligase @16 degree C overnight (since I have both sticky and blunt end)
vector:insert, I have tried 1:5 and 1:10, reaction volume is 30 uL.
(5).Transformation
positive control : uncut vetor only, it works really well
negative control: no colonies
experiment: 2 uL of ligation mixture into 100 uL of JM109 competent cells, electroporation-->1 mL SOC medium-->37 degree C 1 hour-->spread 200 uL culture on LB/Amp plates
NO COLONIES at all

Then I tried top 10 competent cell. I got many colonies. After screening by plasmid minipreps, I did not see band with expected size, however, I saw two bands as well as genomic DNA. Has anybody seen this before?

Here are the controls I have performed.
(1). Long time/sequential digestion
I tried single digestion with SmaI first (overnight),P/C purifying digested product, then digest with NdeI (overnight). Then self ligation was performed. Apparently, there is no difference between short-time digestion and long-time digestion.
(2). Self ligation control
vector: ladder has been observed
insert: ladder has been observed, which includes monomer, dimer, trimer...This implicates both NdeI and SmaI worked, otherwise I should see only monomer and dimer if only one enzyme worked.
(3). Uncut vector was able to transform to JM109 pretty well.


Any comments are welcomed!

Gel:
from left to the right
1kb+ ladder
uncut vector (7.4kb)
miniprep colonies (the expected new plasmid should be 7.7 kb)

I appreciate what seems to be an elaborate description of your technique and problem. however, if you can break it down into two simple questions and then refer to this post for more info, it would be helpful

I think you are working way too hard. The whole point of heat killing enzymes is to avoid having to do a phenol/chloroform extraction. You also should not need to do a gel purification, which also eliminates the likely problem of excess UV exposure of your DNA. The ladder control for your ligation is a really good idea, and pretty convincing. Better is to treat the ligation mixture with the enzymes, and look for the double length product. If you are seeing only double length bands on your insert, one or the other of your enzymes may not be cutting well. The digestion will tell you which one. What kinds of overhangs in your primers are present? Usually you can do the cutting directly in ligation buffer, if a bit of salt is added.

I would try cutting your vector and PCR product with the enzymes, heat killing, then adding them directly to a ligation mix, ligate, and transform. You might have too much background with this strategy. Is there another pTYB2 restriction enzyme between those two which you could cut with? It would help reduce background with this technique.