Calabrese(humanCRISPRa) Library_trouble in amplifying genomic DNA

I just had an issue in amplifying the gemomic DNA from unsorted cells sample. Briefly, I did a sorting-based screen with the Calabrese library(human CRISPRa) and used the Ex-taq protocol mentioned in their paper https://www.nature.com/articles/s41467-018-07901-8, and failed in amplifying the genomic DNA from unsorted cells.

(1 round PCR, 28cycles)

Shown as below the PCR gives very week(but still visible) bands at correct size(495bp).

Lane 1,4 is amplified pDNA library, lane 2, 3, 5, 6 are amplified genomic DNA.

09-10-19-L28S9CalAR1_US_A03.jpg

The cells passed a 7 day's puromycin selection and the control cells(withot virus) all died by day 4, so I believe most of my cells should have the construct.

For this part, my question is: why the amplification is so poor, and how can I improve it?

(So far I tried to double Ex-taq, or use new batch of P5/P7 primers, does not help)

And the second part, I tried to purify the product and sent for QC, the tapstation result looks fine(shown below, major peak is 495bp), however, the qPCR result says only ~30% of my purified product is amplifialble.

QC517.PNG

I'm very confused about this: since the size of the product is correct, they shold be sequencible and can be amplified by qPCR. Does anyone have an idea about this?

Any of your response is appreciated!


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Blonde Goblinabout 3 years ago

Issue 2 has solved: I got a lot of gDNA in my purified product(the size-selection of AMPure did not work very well) and this makes a big difference between qPCR and Quibit.

Now I'm trying Gel-purify and AMPure with diluted PCR product, I will keep update my new QC result.

However, anyone has an idea about the poor PCR amplification?

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