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).

First, 1/10 dilutions of the overnight cultures we made, with LB broth. The Spec was blanked with LB. 1 ml of each sample was loaded into the cuvettes. Background was found to be 48.30 at 600 nm.

'	Sample	OD600	Emission 511	Emission minus background	Corrected emission / OD	'
No	1-No	0.684	82.13	33.83	49.45906433
Med	2-Med	0.691	132.69	84.39	122.1273517
High	3-High	0.674	224.06	175.76	260.7715134
No	4-No	0.735	85.29	36.99	50.32653061
Med	5-Med	0.701	130.51	82.21	117.275321
High	6-High	0.767	228.89	180.59	235.4498044
No	7-No	0.722	84.98	36.68	50.8033241
Med	8-Med	0.795	129.67	81.37	102.3522013
High	9-High	0.721	232.17	183.87	255.0208044
No	10-No	0.747	84.99	36.69	49.11646586
Med	11-Med	0.745	131.97	83.67	112.3087248
High	12-High	0.742	214.47	166.17	223.9487871
No	13-No	0.8	86.45	38.15	47.6875
Med	14-Med	0.757	132.99	84.69	111.8758256
High	15-High	0.715	219.16	170.86	238.965035
Superpos control from David	16-Superpos	1.08	3659.33	3611.03	3343.546296