IGEM:Harvard/2009/Lab Notebook/Week0

Thursday: June 11, 2009

Miniprep of (1) high-GFP, (2) medium-GFP, and (3) GFP-vector. Done to extract DNA for diagnostic and eventual extraction digests, for production of low-GFP; also done to visualize differences in expression levels with different promoters. Followed Quiagen protocol for minipreps. Did three preps of 3 mL ea, (1) high-GFP, (2) medium-GFP, and (3) GFP-vector. Used Nanodrop to look at DNA concentrations, recorded on outside of vials. (1) was approximately 47 ng/uL, (2) was 90 ng/uL, and (3) was 200 ng/uL.

Diagnostic digest of GFP-vector. Done for practice, to see if digest works so we can do a larger scale digest and cut out the GFP and to visualize bands (GFP should be 700-800 bp in size). David made the mastermix, 5 uL GFP-vector DNA (~200 ng/uL) and 10 uL of master mix. Digest with EcoRI and SpeI for 1 hr at 37 degrees.

E-Gel for diagnostic digest of GFP-vector. Done to determine if the digest works. Need to load 20 uL. Only ladder has dye, samples do not need it because gels are buffer free. Prepoured gels. See picture of gel on website. Lane 1—Ladder, Lane 6—Undigested (5uL DNA, 10 uL water), Lane 7—Digested

Use of spectrophotometer to compare expression of GFP under control of different promoters. Cells vortexed and added to cuvettes (make sure they are well mixed). Took OD for each sample and GFP fluorescence at approx 514. Found that all samples (all three, in quintuplicate) had similar OD values. Fluorescence under control of medium promoter was approx 2x basal levels of expression with no promoter. Fluorescence under control of high promoter was approx 2x levels with medium promoter (about 225 for high, 120 for medium, 70 for low; all ODs were about 0.75).