I am new to the use of CRISPR libraries and I would like some input regarding a problem we are having. I am. using the GeCKO v2 library according to the Joung et al. Nature protocols paper.
After library amplification we amplified the sgRNA targets by PCR using an equimolar mix of the primers foward of the paper and one of ...
I am a newfie in this field. To practice before real experiment, I read the nature protocol (DOI: 10.1038/nprot.2017.016) and downloaded python code. To get the test data, I downloaded the fastq file () from the published paper (DOI: 10.1016/j.stem.2018.09.003).
Here, I performed the following code: “ python ./countspacers...
After I amplify the pooled sgRNA library, I need to do next-generation sequencing of the amplified sgrna library to determine sgrna distribution.
So, I do the PCR as step 32 in protocol with 20ng template in 50μl reaction system with 10 reactions in total. The cycle number is 22.
I got 500 μl PCR products and do PCR purif...
Ruby Ghoul
about 3 years ago
Cycles for NGS
I'm following the nature protocol paper, trying to use gecko2 library.
In the paper for the NGS it is suggested to use 80 cycles, but do you think that 75 cycles will be enough?
I will use the NextSeq.
Many thanks
Black Incubus
over 3 years ago
Tapestation shows additional bands
I have been working according to the Joung et al 2017 paper and wanted to ask whether anyone has gotten additional unrelated bands on tapestation?
I pooled the pcr reactions from my amplified library and purified them on Zymo columns. Then I ran 2 micrograms from each sample on an agarose gel at low voltage so as not to he...
Yellow Valkyrie
almost 4 years ago
Gecko mouse library validation
I have a colleague who has just amplified the mouse Gecko library (2 lentivirus system) as per the Joung and Addgene protocols and sequenced on the Miseq to validate coverage. The results have come back as less than ideal.
Skew ratio: 13.5%
% matching guides: 64.2%
% undetected guides: 1.5%
What is the most likely reaso...
Orchid Vampire
about 4 years ago
FASTQ analysis via count_spacer.py question
I am not sure if anyone has tried to do this, but in my lab we decided to design new Reverse primers where we basically replaced the 8 bases index (in the original Reverse primer)
with an Illumina 6 bases index. This change has resulted in our reverse primer to be 2 bases shorter and so we are concern that the count_spacer...
Aquamarine Ghoul
over 4 years ago
Unindexed reads in MiSeq
I did a Gecko library screening following the Julia Joung Nature Protocol paper, but the MiSeq showed that most of the sequences are "unindexed reads".
My primers are the same with the ones in the protocol, and the barcodes are shown in red below:
GeckoR2A| CAAGCAGAAGACGGCATACGAGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTCGCCTT...
Im working with the Gecko v2 library and followed the Joung et al. (2017) Nature Protocols paper very closely.
Now its time fpr NGS.
The company which will sequence the samples asked me if there is special custom primer needed or not?
The second question is: If the PCR Product is around 270 bp and you substract the adapter...
Rose Fenrir
over 5 years ago
sequencing protocols
After library amplification, you recommend sequencing for 80 cycles on an illumina MiSeq. Can you please let me know if you recommend paired end or single end sequencing?
Find your community. Ask questions. Science is better when we troubleshoot together.
Community Guidelines
Sci Find is a place for experimentation beyond the publication. We are a growing collaborative community of scientists and experimentalists from around the world who believe science is better when we work together.
- Help us keep Sci Find a safe space for productive and collaborative conversations. Please be kind and respect your fellow community members. If you see abusive or alarming content, please report it to admin@scifind.net, and our team will take appropriate actions.
- All information posted to this page is public and Open Access. Learn more about our commitment to Open Access by visiting our Docs.
- We encourage our community to have collaborative troubleshooting discussions, and to share tips and tricks, experimental methods, resources, and opportunities.
- You must be a member to create or engage with content. Sign Up to create your profile and join the community for free.
- Follow a guild and create a post. Learn more about guilds here.
- Post titles should be descriptive and include descriptive tags. Check out our tagging guidelines here.