CRISPRCas9 knockout genetic screening: is it necessary to use DMSO in the second PCR?

I trying to set up a CRISPR-Cas knockout screening experiment. I am following two steps PCR protocol, but facing some problems with the sequencing outputs.

In the 2nd Step PCR, is it necessary to use DMSO as the primers are long (~ 100bp) to avoid secondary structures in the DNA template or the DNA primers?

Currently, I am using the following conditions for the 2nd step PCR, but having issues with the sequencing outputs. Please let me know if you also have any suggestions on the following 2nd step PCR conditions.

For a 50 ul reaction, I am using 25ul of the NEB Q5 High-Fidelity 2X Master Mix, 2.5uM Forward and 2.5uM reverse primers, 5ul PCR product from the first PCR, 0.5ul DMSO (1%) and nuclease-free water up to 50ul.

2nd step PCR cycle condition:

Initial Denaturation

|

98°C

|

3 minutes

---|---|---

1 Cycle

|

98°C

|

10 seconds

59°C

|

10 seconds

72°C

|

25 seconds

Final Extension

|

72°C

|

Byron uses 30 seconds

Hold

|

4°C

|


Login or Signup to leave a comment
Fuchsia Kelpiealmost 3 years ago

I have not done this particular 2-step PCR for reading out CRISPR screens before, but I don't usually use DMSO for PCRs even with long primers. In your case, it might be worth doing different numbers of cycles (more than what you are currently doing) on the first and second round PCRs, with different input amounts, and see when you get a band on the gel at the right size.

Find your community. Ask questions. Science is better when we troubleshoot together.
Find your community. Ask questions. Science is better when we troubleshoot together.

Have a question?

Contact support@scifind.net or check out our support page.