Currently in our lab, we are trying to knockdown EGFR gene using Crispr-Cas9 system. We purchased ThermoFisher's GeneArtTM Precision gRNA Kit and followed their user's guide. However, when we are performing the very first step of synthesizing DNA template for gRNA using PCR, we got nearly no product for our desired product (around 110bp), which we already figured that out as we made a mistake when designing our primers. However, we have no idea why our control group (primers provided by ThermoFisher) has two bands when there was only supposed to be one band. We repeated the experiment, this time we even got an extra band in control group compared to the first time, which is super weird. I have attached the pictures of what the band is supposed to look like and our first and second PCR results. The lowest ladder on DNA marker is 100 bp. Can someone share their opinions on this issue? I appreciate it!
Is this happening due to primer dimmer formation? If it is then please go though the following link:
https://www.researchgate.net/post/How_to_avoid_Primer-Dimer_Formation_and_get_our_gene_amplified
For my CRISPR experiments I ended up doing a nested PCR by amplifying my gDNA with primers to make a product of ~500 bp then performing a PCR clean up then doing PCR with internal primers.
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