MLM3636 vector (V012322)

Price Information

Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.

Basic Vector Information

MLM3636 vector Map

Plasmid Protocol

1. Centrifuge at 5,000×g for 5 min.

2. Carefully open the tube and add 20 μl of sterile water to dissolve the DNA.

3. Close the tube and incubate for 10 minutes at room temperature.

4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.

5. Store the plasmid at -20 ℃.

6. The concentration of plasmid re-measurement sometimes differs from the nominal value, which may be due to the position of the lyophilized plasmid in the tube, the efficiency of the re-dissolution, the measurement bias, and adsorption on the wall of the tube, therefore, it is recommended to transform and extract the plasmid before using it

General Plasmid Transform Protocol

1. Take one 100μl of the competent cells and thaw it on ice for 10min, add 2μl of plasmid, then ice bath for 30min, then heat-shock it at 42℃ for 60s, do not stir, and then ice bath for 2min.

2. Add 900μl of LB liquid medium without antibiotics, and incubate at 37℃ for 45min (30℃ for 1-1.5 hours) with 180rpm shaking.

3. Centrifuge at 6000rpm for 5min, leave only 100μl of supernatant to resuspend the bacterial precipitate and spread it onto the target plasmid-resistant LB plate.

4. Invert the plate and incubate at 37℃ for 14h, or at 30℃ for 20h.

5. Pick a single colony into LB liquid medium, add the corresponding antibiotics, incubate at 220rpm for 14h, and extract the plasmid according to the experimental needs and the instructions of the plasmid extraction kit.

MLM3636 vector Sequence

LOCUS       MLM3636.        2278 bp DNA     circular SYN 01-JAN-1980
DEFINITION  CRISPR/Cas9 plasmid for expressing a guide RNA (gRNA) from the U6 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    MLM3636
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2278)
  AUTHORS   Joung Lab / Addgene #43860
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 2278)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..2278
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        82..322
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        350..425
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      426..431
                     /note="polIII terminator"
                     /note="RNA polymerase III transcription terminator"
     rep_origin      complement(529..1117)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(1291..2148)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(2149..2253)
                     /label=AmpR promoter