Beads cleanup after enzyme digestion in RADseq: Yes or no?

Diede · 03-21-2018, 02:10 AM

Hi all,

I am working on double digest RAD sequencing, and I have quite a general question. In the ddRAD protocol of Peterson et al. they do a beads cleanup directly after enzyme digestion. However, I also know people who skip this step and only do the beads cleanup after the ligation. I am curious what you guys do in your lab and why? I suppose it is a balance between increasing the concentration of the DNA versus loss of DNA?

I would like to make a well-informed decision for my samples

Diede

nucacidhunter · 03-21-2018, 02:55 AM

Clean up after digestion is unnecessary. If your adapters disrupts the RE recognition site you just can add ligase, ATP and ligation buffer and continue with ligation. You should choose RE and ligase brand where RE buffer will be compatible with ligation reaction.

If adapters ligation maintains the RE recognition site then RE can be deactivated by heat treatment.

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03-21-2018, 02:10 AM	#1
Diede Junior Member Location: the Netherlands Join Date: Mar 2018 Posts: 2	Beads cleanup after enzyme digestion in RADseq: Yes or no? Hi all, I am working on double digest RAD sequencing, and I have quite a general question. In the ddRAD protocol of Peterson et al. they do a beads cleanup directly after enzyme digestion. However, I also know people who skip this step and only do the beads cleanup after the ligation. I am curious what you guys do in your lab and why? I suppose it is a balance between increasing the concentration of the DNA versus loss of DNA? I would like to make a well-informed decision for my samples Diede

03-21-2018, 02:55 AM	#2
nucacidhunter Senior Member Location: Melbourne Join Date: Jan 2013 Posts: 1,137	Clean up after digestion is unnecessary. If your adapters disrupts the RE recognition site you just can add ligase, ATP and ligation buffer and continue with ligation. You should choose RE and ligase brand where RE buffer will be compatible with ligation reaction. If adapters ligation maintains the RE recognition site then RE can be deactivated by heat treatment.