Team:Duke/Notebook/July

Miniprep 40 cultures of potential crRNA inserts in pdCas9

• Labeled with three numbers
• Date of transformation - crRNA-GFPx - sample number

700uL of each culture saved to freeze in glycerol if colonies appear successful

• Stored at 4C for the day

Analytical restriction digest of potential crRNA inserts

• All 40 tubes + 2 pdCas9 tubes as control
• 1 uL DNA in 10 uL digest for 2 hrs at 37C
• SacI-HF and NheI-HF in cutsmart

Made and ran 1.5% agarose/TBE gels

• To differentiate between small band differences
• Gel 1:
• 1. 2-log ladder
• 2. pdCas9
• 3-12. pdCas9-crRNA-GFP1 from 7/3 transformation
• 13-22. pdCas9 -crRNA-GFP2 from 7/3 transformation
• 23. pdCas9
• 24. 2-log ladder
• Gel 2:
• 1. 2-log ladder
• 2. pdCas9
• 3-12. pdCas9-crRNA-GFP1 from 7/7 transformation
• 13-22. pdCas9 -crRNA-GFP2 from 7/7 transformation
• 23. pdCas9
• 24. 2-log ladder
• Expected results:
• pdCas9 and successful inserts:
• 3.1, 2.8, and 2.7 kb bands, plus 620 bp insert band
• unsuccessful recircularizations:
• 3.1, 2.8, and 2.7 kb bands, plus 585 bp insert band
• Results:

Transformation of CCEC with RFP Construct of varying concentrations+control with no DNA

• Followed Charlie’s Cloning Protocols
• Mistakenly used all of DNA construct from the kit… So not sure if the transformation will be successful
• Plated the transformed DNA and put in incubator at 37C
• Results (6/2/14)--Transformation unsuccessful because improper procedure was done by MFaw

Next Steps:

• Look at plates, compare cultures