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pABS018是一個(gè)植物細胞CAS9和gRNA表達質(zhì)粒。This cloning procedure is very rapid, since the AarI enzyme cuts outside of its recognition site, the overhangs are incompatible and the subsequently-cloned in fragment does not restore the AarI site. Therefore, no purification is required to remove the restriction enzyme prior to ligation. In my experience, more than half the clones contain the desired insert.

Based on: https://groups.google.com/forum/#!msg/crispr/ofvc1KLCMfo/pf7jbjZP9FUJ

Cost, G.J., and Cozzarelli, N.R. (2007). Directed assembly of DNA molecules via simultaneous     ligation and digestion. BioTechniques 42, 84, 86–89.

A. design oligos for cloning

Requirements for a good sgRNA: needs to be 20bp long, followed by NGG. The 20 bp sequence should have as many mismatches as possible with other genes to reduce off-target effects. It should also start with a G (this G may be required for U6 promoter function):

5'-GATTGNNNNNNNNNNNNNNNNNNN3'-        (oligo #1)

      ||||||||||||||||||||

   3'-CNNNNNNNNNNNNNNNNNNNCAAA-5'    (oligo #2)

No need to order oligos phosphorylated so long as you don't de-phosphorylate vector.

*This website is excellent for helping choose a good target site:

   http://www.genome.arizona.edu/crispr/CRISPRsearch.html

B. Anneal oligos to make double-stranded insert with sticky ends:

1. Mix both oligos together in 1x PCR buffer to 10 μM concentration

      20 μL oligo1 (100 μM)

           20 μL oligo2 (100 μM)

           20 μL 10x PCR buffer

          140 μL  H2O

2. Float tubes in a beaker of 100-200 ml boiling water. Allow to cool several hours to room temperature. This ensures that annealing is complete.

C. AarI digest pABS018 plasmid (Concurrently with step B)

      x μL pABS018 (1μg)

      2 μL AarI RE buffer (10x)

      1 μL AarI enzyme (2units/μL)

      y μL H2O (to 20μL final volume)

      →37°C for 1.5 hours

D. Ligate insert + plasmid

   To 20μl digested plasmid, add:

      2.5 μL 10x T4 Ligase Buffer

      1 μL annealed oligo

      1.5 μL T4 DNA Ligase    

      →37°C for 1.5 hours

E. Transform

      Transform 2μL of ligation. Plate on Kanamycin Plates

F. Screen colonies and sequence

Doing colony PCR gives many false positives due to contamination by the ligation reaction. We get around this problem by replica plating the colonies onto new plates, and then doing colony PCR the following day. Alternatively you can PCR flaking the insert and then AarI digest. Those with the insert are AarI-resistant, but background colonies containing the original AarI site are cut by AarI.

ABS 367 GTTGAACAACGGAAACTCGA

ABS 402 GTTTTCCCAGTCACGACGTTG

ABS 405 ATGCAAGCTTGCGGCCGCGCTG

For colony PCR to check for insert (on replica-plated colonies), use ABS367 and your sgRNA reverse primer. This will give a PCR product of ~468 bp.

For colony PCR to flank the insert, use ABS402 and ABS 405. This will give a PCR product of 732 bp. Then sequence that product with ABS 367.

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