IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-8-4: Difference between revisions
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:*The P1 buffer that is in our 4C refrigerator contains more than 700uL of RNase A, which is due to there being more than 700uL of RNase in the container that comes with the miniprep kit. | :*The P1 buffer that is in our 4C refrigerator contains more than 700uL of RNase A, which is due to there being more than 700uL of RNase in the container that comes with the miniprep kit. | ||
==Weight of Perry's Gel segment== | |||
.3260 g | |||
Revision as of 16:36, 4 August 2006
Harvard iGEM is firing on all cylinders (despite missing a few members)...
Ligation
Although the Silver protocol recommends using 50-100ng BB vector + 4-10x insert molar mass, Endy recommends a 3-fold molar excess and less DNA. Roche kit says 1:1 or 1:2 works for DNA which is not similiar in length. Seeing as how our total vector + insert DNA is very low concentration, we will use a 1:1.5 ratio. Additionally, we are adding DNA dilution buffer up to 10uL as the ROCHE protocol says as opposed to Silver's protocol, which says to add water + 2uL DNA dilution protocol up to 10uL.
KaiA
- BB vector: 50ng @ BB backbone high copy 1 (10.6 ng/uL) = 4.7 uL
- BB insert, KaiA: 1.5*(855bp/2000bp)*50ng=32.06 ng @ (10.5 ng/uL) = 3.1 uL
- 1x DNA Dilution Buffer: 2.2 uL
KaiB
- BB vector: 50ng @ BB backbone high copy 1 (10.6 ng/uL) = 4.7 uL
- BB insert, KaiA: 1.5*(300bp/2000bp)*50ng=11.25 ng @ (4.35 ng/uL) = 2.6 uL
- 1x DNA Dilution Buffer: 2.7 uL
Protocol:
- Mix above ratios + 10uL #1 + 2uL #3
- BE sure to mix #1 (ligation buffer) well especially!
- Sit at bench for ~10min
- Put back on ice
Transformation
Did a positive control, negative control, KaiA, and KaiB. Used 15uL of transformant cells on each except for negative (5uL). Used OneShotTop10.pdf protocol off of invitrogen; 5uL of either ligation product, water, or positive control plasmid. 30 min on ice, 30s on 42C. SOC @ 37C @ 1h, plated. The transformed cells were plated and placed in the 37C incubator.
Strains
- We're working with pSB4A3 (this is the low copy plasmid)
- The RFP BioBrick that we had been using is BBa_J04450
Miniprep
Nick grew up the pSB4A3 last night into 7 tubes with 8 mL each of culture. He also had one control, where there was no grown bacteria.
The minipreps were prepared of these cultures.
- The P1 buffer that is in our 4C refrigerator contains more than 700uL of RNase A, which is due to there being more than 700uL of RNase in the container that comes with the miniprep kit.
Weight of Perry's Gel segment
.3260 g
