IGEM:Harvard/2009/Lab Notebook/Week0: Difference between revisions
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=Friday: June 12, 2009= | =Friday: June 12, 2009= | ||
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. | |||
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. |
Revision as of 20:51, 14 June 2009
Tuesday: June 9, 2009
Wednesday: June 10, 2009
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
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.
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.