IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-8-16: Difference between revisions

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* Western blots
* Western blots
** <s>Inoculate several cultures of GFP dev. for Western blotting this afternoon</s>
** <s>Inoculate several cultures of GFP dev. for Western blotting this afternoon</s>
** Take regular measurements of culture OD
** <s>Take regular measurements of culture OD</s>
** Western blot
** Western blot '''(delayed until tomorrow because we don't seem to have GFP)'''
* Construct creation
* Construct creation
** Restreak and do colony PCRs of the Kai\X-P + J04500\S-P transformants to check if our insert is there
** <s>Restreak and do colony PCRs of the Kai\X-P + J04500\S-P transformants to check if our insert is there</s>
** Midiprep the KaiA/B/C in GA plasmid that we grew up from frozen stocks yesterday
** <s>Midiprep the KaiA/B/C in GA plasmid that we grew up from frozen stocks yesterday</s>
*** Digest a large amount of KaiA/B/C\X-P
*** <s>Digest a large amount of KaiA/B/C\X-P</s>
*** Gel-purify
*** Gel-purify
** RBS + KaiC construct
** RBS + KaiC construct
*** Miniprep the B0034 cultures
*** <s>Miniprep the B0034 cultures</s>
*** Digest B0034\S-P
*** <s>Digest B0034\S-P</s>
*** Gel-purify
*** Ligate KaiC\X-P + B0034\S-P
*** Ligate KaiC\X-P + B0034\S-P
*** Transform 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 ==
== Inoculation R0010 + E0241 ==
Line 43: Line 47:


== Midiprep of KaiABC ==
== Midiprep of KaiABC ==
The KaiABC genes were eluted (redissolved) in 200 mL of H<sub>2</sub>O.
:* KaiA: 279.1 ng/uL
:* KaiB: 285.2 ng/uL
:* KaiC: 409.7 ng/uL + 409.5 ng/uL


==Ligation test and replating==
==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.
[[Image: plates_81606.jpg|thumb|right]]


Each reaction:
Each reaction:
Line 52: Line 66:
* colony or 1 uL template or nothing
* colony or 1 uL template or nothing


18 RXNS:
19 RXNS: (18 + 1 pos, which is B0034)
* 8*20=160uL supermix
* 8*20=160uL supermix
* 1*20 = 20uL VF2
* 1*20 = 20uL VF2
Line 65: Line 79:
* 72C for 10'00
* 72C for 10'00
* 4C forever
* 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
[[Image: 1-11_81606.jpg|thumb|left|click for legend]]
[[Image: 12-21_81606.jpg|thumb|left|click for legend]]
<br style="clear:both">
== 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 ==
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Plasmid'''
| align="center" style="background:#f0f0f0;"|'''Enzymes'''
| align="center" style="background:#f0f0f0;"|'''Incubation temperature'''
| align="center" style="background:#f0f0f0;"|'''Incubation time'''
|-
| KaiA in GA||X, P||37C||16 hrs
|-
| KaiB in GA||X, P||37C||16 hrs
|-
| KaiC in GA||X, P||37C||16 hrs
|-
| B0034 in pSB1A2||S, P||37C||16 hrs
|-
| KaiA in GA||X, P||45C||16 hrs
|-
| KaiB in GA||X, P||45C||16 hrs
|-
| KaiC in GA||X, P||45C||16 hrs
|-
| B0034 in pSB1A2||S, P||45C||16 hrs
|-
|
|}
'''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 H<sub>2</sub>O
'''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 H<sub>2</sub>O
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'''
* 10 µL NEBuffer 2
* 2 µL SpeI
* 2 µL PstI
* 1 µL 100x BSA
Pipette 3.75 µL into each reaction.
==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.--[[User:Zsun|Zsun]] 20:31, 19 August 2006 (EDT)

Latest revision as of 17:31, 19 August 2006

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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
click for legend
click for legend


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

Plasmid Enzymes Incubation temperature Incubation time
KaiA in GA X, P 37C 16 hrs
KaiB in GA X, P 37C 16 hrs
KaiC in GA X, P 37C 16 hrs
B0034 in pSB1A2 S, P 37C 16 hrs
KaiA in GA X, P 45C 16 hrs
KaiB in GA X, P 45C 16 hrs
KaiC in GA X, P 45C 16 hrs
B0034 in pSB1A2 S, P 45C 16 hrs

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

  • 10 µL NEBuffer 2
  • 2 µL SpeI
  • 2 µL PstI
  • 1 µL 100x BSA

Pipette 3.75 µL into each reaction.

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)

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