USER troubleshooting guide

If you don't achieve the expected results in your USER experiment, you can check simple things such as storing your plasmids in aliquots smaller than 50 µL or have a look at the suggestions stated below.

X) Result

- Possible cause --> Solution

1) No PCR product

• Product too long --> Split up in two USER reactions

• Wrong polymerase added --> Use Uracil-compatible DNA polymerase

2) No colonies after transformation

• Primers not designed properly --> Double check the primers e.g. with AMUSER

• USER enzyme too old --> Replace enzyme with a new batch. Test enzymes with a cloning reaction that previously worked (can be stored and used as positive control)

3) Few colonies

• Low yield or poor quality of PCR product --> Optimize the PCR for better yield

• Bacteria not competent --> Use a new batch of competent cells or try different types of cells

4) Colonies contain only the template vector

• Template was not properly removed by Dpn I or purification --> Replace Dpn I batch or increase the DpnI incubation time

• Vector self-ligation --> Redesign primers for vector amplification