IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-8-4
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Harvard iGEM is firing on all cylinders
Ligation of KaiA + BB backbone and KaiB + BB backbone
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 of ligated KaiA + bkb and KaiB + bkb in Top10
We transformed our ligated KaiA + bkb and KaiB + bkb from above, along with a positive and negative control (4 transformations total). Used 15uL of Top10 transformant cells on each except for the negative (5uL). Used OneShotTop10.pdf protocol off of Invitrogen; 5uL of either ligation product, water, or positive control plasmid. 30 min on ice, 30s @ 42C. SOC @ 37C for 1h, plated.
The transformed cells were plated and placed in the 37C incubator.
Transformation of J04450 in Top10F'
We found out that our Top10F' competent cells contain lacIq, a gene which overexpresses lac repressor. This is desirable because it supposedly makes the Lac promoter less leaky.
To test the effectiveness of lacIq, we transformed our J04450 plasmids in Top10F'. If all goes well, the transformant cells should show little or no constitutively-on fluorescence.
We did 3 transformations:
- 5 µL J04450 (nanodropped at ~20 ng/µL), 15 µL Top10F' cells
- 5 µL H2O, 15 µL Top10F' cells
- 1 µL positive control (plasmids provided by Invitrogen), 15 µL Top10F' cells
Incubated for 30 min on ice, heat shocked @ 42C for 30 s. Mixed with 250 µL SOC and plated.
4 plates:
- Negative control
- Positive control
- J04450 #1
- J04450 #2
Strains
- We're working with pSB4A3 (this is the low copy plasmid)
- The RFP BioBrick that we had been using is BBa_J04450
Miniprep of psB4A3 (low-copy plasmid)
Nick grew up the pSB4A3 (low-copy plasmid) last night into 7 tubes with 8 mL each of culture. He also had one control, where there was no grown bacteria.
We miniprepped the 7 cultures, which should be sitting in the "bundles of joy" container in the 4C.
- 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.
Digest of J04450 with XbaI and PstI
We performed another digest of BBa_J04450 with XbaI and PstI, since our first one appeared to fail:
The reaction was:
- 19.25 µL DNA
- 2.5 µL Buffer 3 (from NEB)
- 0.25 µL BSA (100x) (1µL of BSA diluted in 3 µL of H2O, and 1µL of this mixture)
- 0.5 µL XbaI enzyme
- 0.5 µl PstI enzyme
- 2 µL H2O
The digest schedule is 5 hours @ 37C, 20 min @ 80C, hold at 4C.
Inoculation of J04450
Since we're nearly out of our miniprepped J04450, and we'll want more backbone if our current ligation fails, we inoculated a new culture from our frozen stock. 8 mL LB, 8 µL Amp (at 50 mg/µL).
Perry's Gel segment
Weight: 0.3260 g
The gel segment was excised and purified, and now resides in "P.O. box" PTSA13
