Issues with NGS Library Prep

pi-rat
2023-12-08T11:02:59Z

Greetings Folks, I apologize if this is the wrong spot for this but I'd like to cast a net to see if I can get any additional thoughts or help.

I recently started a new job as NGS Library Prep Tech - sadly I had only begun training on this briefly at my last position but only got an introduction to Speed/Mag Bead clean up. I was hired because the lab is growing quickly and has had issues with organizational stuff in the past and that is my strong suit (my last position I did a lot of clonal DNA / miniprep stuff as far as the wet work went).

The person I was replacing at my new job was only there for two days and didn't really help a whole lot other than hand me a haphazardly written protocol and said "practice by cleaning ladders at different bead concentrations and running them on a gel."

Did that and was told they look good.

Fast forward to using actual samples: There were a set that needed to be redone because the final pool was lost. When I did my first qubit quants after the post PCR speed bead clean up I noticed that the quant concentrations were ~80% less than what they had been previously.

Today I have some remaining sample that I can push through PCR, my plan is to quant 8 out of the 53 samples of the pre / post PCR plate and then again after I do clean up.

As far as clean up goes I had been trying to do the whole plate at once, but I'm going to go back to just completing a column at a time to ensure my timing is better.

Are there particular spots in the Speed / Mag Bead clean up process that I should be aware that I could be washing away / losing DNA?

Do people have any tips on how to be more 'sure footed' in this process?

Ways that I can better practice and say "yup, I've got this!"?

Thanks for the help, and if this should be posted somewhere else please let me know, but as this is 'science' related I thought it fit best here.

Comments URL: https://tildes.net/~science/1cqo/issues_with_ngs_library_prep

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