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:
• Gel 1 appears to only be recircularized
• Gel 2 has some promising inserts:
• GFP1 samples 1,2, and 3 from 7/7 transformation
• GFP2 samples 7,8, and 9 from 7/7 transformation
• Glycerol stocks frozen for these six samples
• Next Steps:
• Run digest with BsaI to confirm that plasmids are not uncut pdCas9
• Sequence to confirm presence of insert (using oligos ordered 7/8/14)

Miniprep 3 new backbones

• pSB6A1-J04450 (3 copies)
• concentrations 437.6, 483.9, and 450.1 ng/uL
• pSB3K3-I20260 (3 copies)
• concentrations 114.6, 120.0, and 97.3 ng/uL
• pSB4K5-J04450 (3 copies)
• concentrations 129.3, 165.9, and 155.1 ng/uL

Prep-scale digest of all 9 backbone tubes

• 50 uL DNA in 60 uL total reaction
• EcoRI-HF/Spe-HF in cutsmart
• 3 hours at 37C

Agarose gel extraction of 3 backbones

• Gel 1, Left: pSB6A1
• Gel 1, Right: pSB3K3
• Gel 2, Left: pSB4K5
• All 3 lanes had 2 bands as expected
• upper band (backbone) in 3K3 had messy trail behind it (not extracted)
• All 180 uL from three tubes combined with 20 uL loading dye in x-large gel well
• top band extracted from all 3 plasmids, split into 2 tubes each, stored at -20C

Streak plate from frozen stock

• pSB1C3-K608012
• In order to switch into pSB6A1

Transformation from iGEM distribution kit plates

• Plate 2-6D: pSB1C3-BBa_R0011
• Engineered Lac promoter (not affected by glucose)
• Plate 3-15O: pSB1C3-BBa_I13500
• Strong RBS-GFP
• Plated on LB+Cm

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