pML107酵母基因编辑CRISPR-Cas9载体-BioVector NTCC质粒载体菌种细胞蛋白抗体基因保藏中心

pML107

BioVector NTCC质粒载体菌种细胞基因保藏中心                      

Expresses Cas9 and contains guide RNA expression cassette with BclI-SwaI cloning sites for guide sequence cloning; Contains LEU2 marker for yeast transformation
Recovery
1. Obtain an LB agar plate with the appropriate antibiotic.
2. Using a sterile pipette tip, touch the bacteria growing within the punctured area of the stab culture. (A sterilized wire loop or sterile toothpick can be used in place of a sterile pipette tip.)
3. Run this tip lightly over a section of the plate, as shown in the figure, to create streak #1.
4. Using another sterile pipette tip, pass through streak #1 and spread the bacteria over a second section of the plate, to create streak #2.
5. Using a third sterile pipette tip, pass through streak #2 and spread the bacteria over the last section of the plate, to create streak #3.
6. Grow overnight in a 37 o C incubator (unless a different growth temperature is indicated on the plasmid datasheet).
7. In the morning, single colonies should be visible. If the bacterial growth is too dense, re-streak onto a new agar plate to obtain single colonies.


• Vector backbone
pRSII425
• Backbone size w/o insert (bp)6841
• Total vector size (bp)12366
• Vector type
Yeast Expression, CRISPR
• Selectable markers
LEU2
• Bacterial Resistance(s)
Ampicillin
• Growth Temperature
37°C
• Growth Strain(s)
DH5alpha
• Growth instructions
Need to purify DNA from Dam- E.coli strain for efficient BclI cutting
• Copy number
High Copy
• Gene/Insert name
Cas9
• Species
S. cerevisiae (budding yeast); Streptococcus pyogenes
• Insert Size (bp)
4151
• PromoterpTDH3
• Cloning methodRestriction Enzyme
• 5′ cloning siteEcoRI (not destroyed)
• 3′ cloning siteXhoI (not destroyed)
• 5′ sequencing primerT7
• Gene/Insert name
single guide RNA expression cassette
• Species
S. cerevisiae (budding yeast), Synthetic
• Insert Size (bp)
447
• PromoterpSNR52
• Cloning methodRestriction Enzyme
• 5′ cloning siteSacII (not destroyed)
• 3′ cloning siteEagI (not destroyed)
• 5′ sequencing primerT3

BioVector NTCC质粒载体菌种细胞蛋白抗体基因保藏中心
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