IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-8-16

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To-do

 * Western blots
 * Inoculate several cultures of GFP dev. for Western blotting this afternoon
 * Take regular measurements of culture OD
 * Western blot (delayed until tomorrow because we don't seem to have GFP)
 * Construct creation
 * Restreak and do colony PCRs of the Kai\X-P + J04500\S-P transformants to check if our insert is there
 * Midiprep the KaiA/B/C in GA plasmid that we grew up from frozen stocks yesterday
 * Digest a large amount of KaiA/B/C\X-P
 * Gel-purify
 * RBS + KaiC construct
 * Miniprep the B0034 cultures
 * Digest B0034\S-P
 * Gel-purify
 * Ligate KaiC\X-P + B0034\S-P
 * Transform KaiC\X-P + B0034\S-P

Miniprep of B0034
Title says it all; 2 60uL samples of B0034 in the freezer, and glycerol stock.

Inoculation R0010 + E0241
10 inoculations of the R0010 + E0241 were made:


 * 10 mL LB amp & 1 ul R0010 + E0241
 * 10 mL LB amp & 1 ul R0010 + E0241 (IPTG - not yet added)
 * 10 mL LB amp & 8 ul R0010 + E0241
 * 10 mL LB amp & 8 ul R0010 + E0241 (IPTG - not yet added)
 * 10 mL LB amp & 64 ul R0010 + E0241
 * 10 mL LB amp & 64 ul R0010 + E0241 (IPTG - not yet added)
 * 10 mL LB amp & 256 ul R0010 + E0241
 * 10 mL LB amp & 256 ul R0010 + E0241 (IPTG - not yet added)
 * 10 mL LB amp & 4.1 mL R0010 + E0241
 * 10 mL LB amp & 4.1 mL R0010 + E0241 (IPTG - not yet added)

Midiprep of KaiABC
The KaiABC genes were eluted (redissolved) in 200 mL of H2O.


 * KaiA: 279.1 ng/uL
 * KaiB: 285.2 ng/uL
 * KaiC: 409.7 ng/uL + 409.5 ng/uL

Ligation test and replating
We had 18 colonies grow up; see the image at the right. For each of these, I plated and did a colony PCR to test for the insert. For the first 8, I also innoculated, though it may not work.



Each reaction:
 * 8 µL PCR Supermix
 * 1 µL VF2 primers @ 2uM
 * 1 µL VR primers @2uM
 * colony or 1 uL template or nothing

19 RXNS: (18 + 1 pos, which is B0034)
 * 8*20=160uL supermix
 * 1*20 = 20uL VF2
 * 1*20 = 20uL VR

PCR schedule:
 * 95C for 15'00
 * Do 30 times:
 * 95C for 0'30
 * 55C for 0'30
 * 72C for 2'00
 * 72C for 10'00
 * 4C forever

Results:

I'm confused.... the + control is B0034 (the RBS), for reference; the actual coding sequence is i think 33bp or something really short, so basically add on the size of the band in the gel to the expected KaiA,B,and C sizes.

EDIT: Actually, the previous ligation was not a self-ligation like thought. Look at http://www.openwetware.org/wiki/Image:2006_08_14_egel2.jpg, we expect a 550 bp band (like + control) if it self-ligated but it actually is a 300bp band...

EXPECTED
 * KaiA from VF to VR2: 855bp + 536bp (VF2-VR on J04500) - 6bp+20bp (prefixes) = 1.4kb
 * KaiB: 309bp + 536bp (VF2-VR on J04500) - 6bp+20bp (prefixes) = 859bp
 * KaiC: 1560bp + 536bp (VF2-VR on J04500) - 6bp+20bp (prefixes) = 2110bp
 * B0034 Positive: 250bp (VF2-VR) = 250bp
 * Nothing: 536bp





Innoculation of GFP Device from Nick's Plate
4 Innoculations were made of GFP (R0010 + E0241) from Nick's bright green plate. The cells could be dead, though, so no guarantees. All 4 innoculations were made in 10mL LB + Amp. Also made a negative control of 10mL LB + Amp.

Digest of Kai\X-P and B0034\S-P
Kai\X-P reactions
 * 8 µL midiprepped DNA
 * 2.5 µL NEBuffer 3
 * 0.5 µL XbaI
 * 0.5 µL PstI
 * 0.25 µL 100x BSA
 * 13.25 µL H2O

Kai\X-P master mix
 * 17.5 µL NEBuffer 3
 * 3.5 µL XbaI
 * 3.5 µL PstI
 * 1.75 µL 100x BSA
 * 92.75 µL H2O

Pipette 17 µL into each reaction.

B0034\S-P reactions
 * 21.25 µL DNA
 * 2.5 µL NEBuffer 2
 * 0.5 µl SpeI
 * 0.5 µL PstI
 * 0.25 µL 100x BSA

B0034\S-P master mix Pipette 3.75 µL into each reaction.
 * 10 µL NEBuffer 2
 * 2 µL SpeI
 * 2 µL PstI
 * 1 µL 100x BSA

Digest of J04500 /xp and /sp
Did 3 digests of J04500 /xp and /sp ea., in 10uL of sol'n as following Silver protocol. 45C for 10h.--Zsun 20:31, 19 August 2006 (EDT)