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. Excitation was set to 488 nm and emission was set to 511 nm. 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 nm	Emission 511	'
Med	0.232	51.58
High	0.261	57.75

'	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

Friday: June 12, 2009

Outlined plan for today: 1. Digest of GFP and vector 2. Gel purification by using either (a) The filter paper method (better yield) (b) Qiagen Gel Purification Kit (contaminants go in column which might obscure spec results, but faster, with this method the gel gives nice results but the spec sometimes gives negative values)

Minipreps of psB3K3 to extract. Anu and Amy did minipreps of my 4 mL cultures of psB3K3 transformed bacteria (name of strain?), and set up my digests. Digested with SpeI and EcoRI.

DNA spec results (values in concentration units if mg/microliters) XG low: 73.6 IB low: 38.2 AR low: 30.4 AG low: 91.3 NK low: 73.6 GD low: 92.0

Double digest protocol: Restriction enzymes: EcoRI + SpeI (New England Biolabs) Common digestion ingredients: (1) Enzymes - to cut the DNA and catalyze the "digestion" reaction (2) DNA - the digestee (3) Buffer (10x)- provides necessary nutrients to run the reaction (4) BSA - adds extra protein/enzyme cushioning which simulates the natural enzymatic environment, chemically innocuous (5) H20 - get up to final volume

GFP insert digest 30 microliters total volume (DNA in EB buffer) We have 17 microliters of DNA left since 2, 5, and 5 microliters of our stock was used up in the spec, digest sample, and undigest samples, respectively

This is our digest suspension (30 microliters total) 17 plasmid in EB 3 buffer (EcoR1 buffer: we are using this buffer becuase since we are doing a double digest, these are complimentary and the buffer works for both enzymes. Usually the reason why we are forced to do single separate digests is because of buffer incompatibility) 3 BSA 1 EcoR1 1 Spe1 5 H20

14x volume (we have 11 tubes)

So in total we have: 42 EcoR1 Buffer 14 Spe1 14 EcoR1 42 BSA 70 H2O

Making agarose gel for extraction of GFP from vector (GFP-vector). Ethidium bromide—0.5uL per mL Medium size gel tray holds 150 mL of agarose, when making 1% gel, 1.5 g agarose and 150 mL TBE. When making a gel use the same buffer to make the gel and as the buffer. Microwave for about 1-2 minutes, cap not tight, careful about sudden boiling, do not reboil after add ethidium bromide. Do not use short wave UV to visualize DNA, it is mutagenic. Poured two gels and ran out GFP-vector digest on one and pSB3K3 on the other one. When imaged using long wave UV found that GFP fragment was right size (about 700) and use filter paper extraction to get DNA. The psB3K3 band was the wrong size, expected about 2.35 kB, but got about 7 kB.

The filter paper extraction is much much more efficient, maybe 5/6, whereas with Quiagen best is going to be 1/10 extraction. After nanodropping, we found the concentration of the DNA to be around 8.6 ng/uL.

We also resuspended our primers.

On Monday we plan to assemble the low promotor and run a diagnostic gel.