Sometimes you can not see your construct on gel. This could because of many reasons. For cloning confirmation I recommend you transform the vector into a Ecoli and culture in selective condition after ligation. You know that linear plasmid can not express in cells and colony will not forms. so after overnight culture any formed colonies are the colonies contain your circular plasmid. for insert confirmation pick up the colony, drop onto a PCR tube directly and perform PCR with the insert gene primer pairs. dont forget perform one cycle 96 C for 10 min as pre-heating for bacterial lysis and release DNA before PCR main cycles.

Good luck

We usually use the Promega pGEM Kits to clone our products for M13 sequencing. It works every time and we can see the bands on the gel (approx 100bp). The kit comes with ligase, a vector, a positive control, and if you want, also the bacteria. It's quite quick to do and reliable.

You could ask for a free sample from the company to try it on your product and then decide if it's ok for you.

This is the protocol(roughly) we use to ligate inserts into vectors using SmaI sites. It works well for us.

setup a SmaI digest with no more than 200 ng/ul (SmaI gets choked up by too much DNA). Place reaction in either 25C water bath or room temperature water bath, allow to set overnight. The next day using a fresh PCR reaction, set up your ligation using the digestion reaction as the template in a 1:5 and 1:10 ratio vector:insert. Then place the reaction in a 4C water bath or and water bath placed in the refrigerator and allow to sit for 8-12 hrs (~overnight). Then transform 3ul of ligation reaction into 100ul electrocompetent cells. recover and grow in rich media for 1 h. then plate dilutions onto a selection media, to get single colonies. Incubate until colonies form. Then either the insert has the selection marker or you perform colony PCR with primers specific for the insert.

I use SmaI to cut my plasmid (pUC). After that I use Blund End repair kit for my PCR product. Then, I made the ligation with T4 DNA Ligase (16oC-2h). On agarose gel, I don't get any band, even for the PCR product. After the repair, I clean the product, but the amount is as the initial, so I don't believe that my product is missed. Any idea why this happened?

Biancastella Cereser when you use these kits you use the M13 vector ( or any other) instead of the vector provided with the kit?

The vector provided by promega (just google promega Vector pgem easy to see the map in their pdf) has got the sequence for M13 -both forward and reverse- in the insertion site, so when you insert your DNA, M13 will automatically get tagged to it. You can then use M13 to sequence your insert after the cloning. We always use M13 to sequence our inserts.

Thank you!

Panagiotis,

Let me try to understand your question - You end repair the PCR product, digest the M13 vector with SmaI, purify and ligate. Sometimes, it is hard to see ligated DNA on agarose gels. I would suggest that you transform E.coli and check ligation efficiency.

You say that you do not see anything on the gel after end repair. Is it possible that you have low levels of DNase activity in the end repair kit? Mix some other DNA (1 or 2 ug) with the kit components and incubate under the same conditions and check on a gel.

Thank you all. I'll try also with PEG addition,due to blund ends.