Mike Barnkob:Protocols/CRISPR/Screening for inserts
From OpenWetWare
Jump to navigationJump to search
Screening for inserts
Basic steps for screening for inserts.
Reagents
- Transformed bacteria
- Sterile pipet-tips.
Protocol
Colony PCR and liquid cultures:
- After having transformed bacteria, pick 5-10 colonies from each plate with pipet-tip.
- ...
Miniprep good colonies:
- [[Mike_Barnkob:Protocols/Bacterial/Isolate_DNA|Isolate plasmid DNA from transformed bacteria].
Send DNA to sequecing:
Check sequences: Go through these basic steps to ensure that the transformed bacteria have the correct alignment and contain the right construct:
- Check sequencing quality in FinchTV:
- Search for gRNA sequence.
- Check quality values: you want to see good chromotographic values as compared to background.
Note: Beginning of data is usually of low quality.
- Copy sequence to SnapGene:
- Find the following features:
- Top strand of gRNA oligo
- gRNA scaffold: GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT
- Filler between gRNA scaffold and EFS: GAATTCGCTAGC
- EFS: TAGGTCTTGAAAGGAGTGGGAATTGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGATCCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGG
- Filler between EFS and Cas9: ACCGGTTCTAGAGCGCTGCCACC
- Beginning of Cas9: ATGGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAG
- Check that extra bases have not been inserted between features.
- Align sequence against backbone sequence.
Note: The beginning and end of the sequence is often of low quality, so pay most attention to the sequences after the first 100bp or so.
- Find the following features:
References
Programs:
- Geospiza FinchTV program: http://www.geospiza.com/Products/finchtv.shtml
- Serial Cloner: http://serialbasics.free.fr/Serial_Cloner.html
