IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-8-10
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Ligation Attempt #2 of J04450+PSB4A3 (see Monday)
Previous ligation attempt did not survive the resistance innoculation. Due to the low amounts of the ligation product, will redo experiments but allign molar amounts as per Endy protocol instead of using a fixed amount like last time:
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 1:2 ratio.
Ligation
- BB vector pSB4A3: 50ng @ BB backbone high copy 1 (3.2 ng/uL) = 15.625 uL
- BB insert J04450: 2*(~1069bp/3339bp)*50ng=24.01 ng @ (6.0 ng/uL) = 5.33 uL
- 1x DNA Dilution Buffer: 4 uL
Protocol (for this only because it is above 10uL):
- Mix above ratios + 25uL #1 + 2.5uL #3
- BE sure to mix #1 (ligation buffer) well especially!
- Sit at bench for ~30min
- Put back on ice
Transformation
For the transformation I did 6 samples:
- 1 exp. Top10 (50uL), Positive control, negative control (25uL ea.)
- 1 exp. Top10F (50uL), Positive control, negative control (25uL ea.)
Ice incubation 30m, 30s@42C, 2m@ice, SOC@1h@37C. Plated on LBCarb.
NOTE: I could of sworn i put SOC, though I found another thing of SOC in the heat plate which makes me question myself... has anyone else done transformations recently?
NOTE: Just in case I added 250uL more SOC 30 min into the incubation and created a duplicate set of plates. I will incubate a total of 1h30 and plate on two and pray it works =D
Results
Checked it today at 10PM; no growth on the experimental but the + control is littered with colonies; will keep in o/n again but probably didn't work. Alain said Kan resistance with the top10 has had trouble, and since the thing is low copy we may not have enough dna for the top10 to work; we can use xls1 instead and try it again.--Zsun
Re-run of gel from Tuesday, w/ undigesed... + gel purification of \XP KaiA
I gel purified the samples 2, 4, and 7; these are marked with a yellow sticker "Kai A \XP 8.11.06" in the Bundles of Joy" box in the cyanobacteria freezer.
Egel attempt of visualizing ligation products pre-transformation
Apparently ligation products don't visualize at all on gels (usually the ligation is transformed and plated ... etc., before it can be visualized; thus, having no products does not signify that the ligation did not work). =(
==Notes from peng==
Today, we should miniprep in the morning the J04550 vector (I think that s what its called) which we innoculated yesterday, and then proceed cutting that with SP and ligating it into the KaiA \XP I prepared above - this will make our first workable construct if all goes well =D Also, the assays should be done, though prehaps we should make doing the above step a priority.
I may be in a bit later today, depending if I get out before daylight :D
To-do
- KaiA + J04500 construct
Make frozen stocks of the J04500 transformants we grew up yesterdayMiniprep (10 mL total volume)Digest J04500\S-P- Ligate J04500\S-P + KaiA\X-P
- Digest assay and Western blot tutorial prep
Miniprep the R0010 + E0241 transformants we grew up yesterday (R0010 + E0241 is hereafter referred to as "GFP device") (8 mL total volume)Perform digest assay on GFP device plasmids and pSB4A3 low-copy plasmid (which we've previously miniprepped)
Digest Assay
Since we lacked a little bit of the GFP device (R0010&E0241), we added 50 uL of ddH2O, and then this was added to Master Mix 3 and 4.
Since there still was not enough of the GFP device(R0010&E0241), we added an additional 20 uL of ddH2O, and used for Master Mixes 1&2
Everything was added according to the assay (on the table from 8/9), but the enzymes were left out for about 15-20 minutes on the bench and on ice. This may affect the digestions (16hrs was digested first, then 12 hrs, then 4 hrs, then 6 hrs, and finally 2 hrs). Each of the digestions were in their own separate PCR machine, except for the 2 hr digestions which were in a water bath. This was timed.
Along with this was J04500 /SP in 16h.
