Are you using TA clone? If yes, make sure the PCR product will have A at the end, which is dependent on the used PCR polymerase. Some one will not generate A at the end of the product.  

I would try to use more genetic material. For that plasmid and large inserts I find that using about 100 ng vector to 300 ng insert, a 3:1 ratio, works the best. Use a 20 uL ligation reaction and incubate for 1 hour at room temperature. Add the entire mixture to 50 uL of chemically competent cells on ice and begin the heat shock procedure. After, add 500 uL of LB broth or sock media and incubate with shaking at 37C for 1 hour before centrifugatiin at 8,000 RPM for 5 minutes, remove 450 uL of supernatant and resuspen do cells in the remaining ~ 100 uL. Plate undiluted onto agar with antibiotic selection. Hope you get your clones and good luck!

I am not using Topo TA, and the primers I designed have the appropriate overhangs to bind the Topo vector. Thomas, how do I make a 20 ul ligation reaction if the original recipe from invitrogen is only for 6 ul (.5-4 ul insert + 1 ul Topo vector + 1 ul salt solution + MilliQ water to volume of 6 ul)? And my second question is do I add the full 20 ul ligation reaction to 50 ul competent cells for a total volume of 70 ul? Lastly, do you think there is any benefit from using the provided company made competent cells rather than my homemade competent cells? Thank you for your suggestions I am going to give this a try today!

I think Thomas's protocol may work. Have you checked the efficiency of your competent cell. it is best to used the commercial one. 

Hi Emily,

In concordance with the advice above, I would suggest

1. Use alkaline phosphatase treatment on the restriction digested vector to eliminate self ligation.

2. Incubate ligation reactions overnight.

3. After you mini prep selected transformant colonies, do a double digest to screen for which colonies carry both the insert and your pET.

Cheers

Please contact www.aas.inc.co. They have a number of very competitively priced high quality cloning kits and would be happy to give you free samples

You probably thought of all of this, but just in case it's still not working, consider:

1.  Even with the sequence for directional cloning in your primers, the kit you are using requires blunt PCR products.  Make sure you are using a proofreading Taq so the A's are removed.  Also, make sure there is a 10 minute 72 degree step at the end of the PCR.  Filling in the last few bases and proofreading, which needs to be perfect for cloning, can be much slower than most of the amplification.

2.  Are you sure the clones you got are not the original plasmid that was the template for PCR?  If you don't gel purify, you should get this back unless it has a different antibiotic resistance gene than your target vector.

3.  Definitely try the competent cells that come with the kit.  I think there might be some magic to these cells that Invitrogen worked out for Topo cloning.

I also use same kit  pET directional TOPO expression  kits. If you do not use a proofreading polymerase your insert may not be cloned. Just as suggested by Michael. I have initially used a non proofreading polymerase but do not get my GOI inserted

Hi Emily J Shultz, 

I have the same questions as you so I was wondering how the experiments are going for you? Whats your experience of larger ligation reaction volume as suggested by Thomas Weppelmann? And have you tried different kits?

Best regards,

Sanna Gudmundsson