IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-8-15

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Contents

To-Do

  • Reinoculation of GFP device.
  • Inoculation of B0034 from plate.
  • Watch good Ivy player (courtesy of Jeff)
  • Gel purification of KaiABC from yesterday
  • Run gel of digests from this morning
    • Gel purify good bands
    • Ligation of KaiA/B/C\X-P into J04500\X-P and J04500\S-P
    • Transformation
  • Inoculate KaiABC for a midiprep
  • Run Western Blot (postponed for tomorrow)

Yet another digest attempt

From our previous gel, it can be argued that the J04500 digest was inconclusive and that the KaiA,B,C digests were poor. Looking back on previous results, the first digest I ever did seemed to be the cleanest one run and gave a working ligation; I'm posting the results of that digest below. The protocol is virutally the same though and we redid this digestion a couple of days ago at varying time pts.... what could cause the diff. results?

Here is a digest attempt using 45C: http://www.neb.com/nebecomm/products/faqproductR0133.asp#850 says it causes an increase in activity...

What Dave and I did before (6/14)

  • Digestion of vector/insert
    • Digested R0010 (200bp cutout) as vector at S and P site.
      • .5uL Spe1, .5uL Pst1
      • 11uL h20
      • 2.5uL 10X BSA
      • 2.5uL #2 NebBuffer
      • 8uL DNA
    • Digested other 2 (~900bp cutout) as insert at X and P site.
      • .5uL Xba1, .5uL Pst1
      • 11uL h20
      • 2.5uL 10X BSA
      • 2.5uL #3 NebBuffer
      • 8uL DNA
    • Incubate @ 37C for 1h
  • Phosphatase
    • 80C@15min to kill enzyme activity
    • Used CIP (1 unit) into the R0010, 1h@37C
  • Run on 1% agarose gel
  • Image, Cutout, and Purify
    • Can isolate the three from the gel

Result

"results of PCR"
"results of PCR"


 Ladder=1kb+
 Lane 1=R0010 (#1)
 Lane 2=E0241 (#2)
 Lane 3=E7104 (#3)

Protocol

Label DNA Amount Water Enzymes Amount NEB buffer BSA (100x) Time Master Temperature
Digests1J0450019.5 µL1.75 µLXbaI & PstI0.5 µL, 0.5 µLBuffer 3 (2.5 µL)0.25 µL 2 hrs 1 45C
2J0450019.5 µL1.75 µLSpeI & PstI0.5 µL, 0.5 µLBuffer 2 (2.5 µL)0.25 µL 2 hrs 2 45C
3J0450019.5 µL1.75 µLXbaI & PstI0.5 µL, 0.5 µLBuffer 3 (2.5 µL)0.25 µL 6 hrs 1 45C
4J0450019.5 µL1.75 µLSpeI & PstI0.5 µL, 0.5 µLBuffer 2 (2.5 µL)0.25 µL 6 hrs 2 45C
5KaiA3uL stock + 16.5uL h201.75 µLXbaI & PstI0.5 µL, 0.5 µLBuffer 3 (2.5 µL)0.25 µL 6 hrs 1 45C
6KaiB3uL stock + 16.5uL h201.75 µLXbaI & PstI0.5 µL, 0.5 µLBuffer 3 (2.5 µL)0.25 µL 6 hrs 1 45C
7KaiC3uL stock + 16.5uL h201.75 µLXbaI & PstI0.5 µL, 0.5 µLBuffer 3 (2.5 µL)0.25 µL 6 hrs 1 45C
Master Mixes1 - - 10.5 µLXbaI & PstI3 µL, 3 µLBuffer 3 (15 µL)1.5 µL - -
2 - - 10.5 µLSpeI & PstI3 µL, 3 µLBuffer 2 (15 µL)1.5 µL - - 22 µL

Pipet 5.5 into ea.

Running in PCR #6 and the thermocycler in the gel room.

Notes for Jeff:

Look on yesterday for the results of running J04500 longer; the undigested band didn't run as fast as the others, so I don't quite trust that gel... it's sitting in the fridge at our bench if you want to extract anything. Sorry I didn't get a change to gel purify the KaiA,B,C, though 6 tubes with the gel pieces are sitting in the green holder in our fridge; if you guys have time, try to purify it today. Otherwise, let's see how this digest runs; and we will definately need to do a ligation for J04500 with our gel purified KaiABC tonight.

GFP device issues

The GFP device we inoculated yesterday, from Peng and David's R0010 + E0241 ligation at the beginning of summer, does not appear green. The frozen cultures was probably picked from a colony that didn't express GFP. Nick has provided us with a working frozen stock, which we inoculated at around 1PM in preparation for our Western blots.

Gel purifications of KaiA/B/C\X-P from last night's digest

I purified the KaiA/B/C\X-P that Peng extracted from last night's digest. Eluted in 50 µL H2O. The nanodrop readings are better than our earlier gel purification of KaiA/B/C\X-P: about 36 ng/µL for KaiA, 15 ng/µL for KaiB, and 100 ng/µL for KaiC. 'EDIT: actually, the numbers were off; redid the nanodrop with 2uL and got better (albeit lower) numbers.--Zsun 19:24, 15 August 2006 (EDT)

Image of Peng's late-night digest

"click for legend"
"click for legend"

It looks like our J04500 digested cleanly, except for J04500\X-P 2 hrs. We will extract and purify J04500\X-P 2 hr, J04500\S-P 2 hr, and J04500\S-P 6 hr.

Inoculation of KaiA/B/C in GA plasmid for midiprep

KaiA/B/C in GA plasmid were inoculated from frozen stock in 25 mL LB amp and placed in the shaker at 6:10 PM.

Inoculation of B0034 transformants

We made 4 inoculations of the B0034 transformants: 2 from each plate. 5 mL LB-Amp.

Ligations for construct making: J04500\X-P + KaiA/B/C\X-P and J04500\S-P + KaiA/B/C\X-P

We performed six ligations, ligating each of KaiA/B/C\X-P into J04500\S-P (for our experimental constructs) and J04500\X-P (to make a proper BioBrick suitable for submission to the registry).

Trying:

  • J04500 /XP + KaiA /XP digested today and yesterday, respectively.
    • BB vector: 50ng @ (6.6 ng/uL) = 7.58 uL Scaled: 5.39uL"'
    • BB insert: 3*(~900/2079bp)*50ng=64.93 ng @ (10 ng/uL) = 6.49 uL Scaled: 4.61uL"'
  • J04500 /XP + KaiB /XP digested today and yesterday, respectively.
    • BB vector: 50ng @ (6.6 ng/uL) = 7.58 uL Scaled: 4.74uL"'
    • BB insert: 3*(~350/2079bp)*50ng=25.25 ng @ (10 ng/uL) = 8.41 uL Scaled: 5.26uL"'
  • J04500 /XP + KaiC /XP digested today and yesterday, respectively.
    • BB vector: 50ng @ (6.6 ng/uL) = 7.58 uL Scaled: 2.91uL"'
    • BB insert: 3*(~1700/2079bp)*50ng=122.65 ng @ (10 ng/uL) = 18.5 uL Scaled: 7.09uL"'
  • J04500 /SP + KaiA /XP digested today and yesterday, respectively.
    • BB vector: 50ng @ (5 ng/uL) = 10 uL Scaled: 6.06uL"'
    • BB insert: 3*(~900/2079bp)*50ng=64.93 ng @ (10 ng/uL) = 6.49 uL Scaled: 3.94uL"'
  • J04500 /SP + KaiB /XP digested today and yesterday, respectively.
    • BB vector: 50ng @ (5 ng/uL) = 10 uL Scaled: 5.43uL"'
    • BB insert: 3*(~350/2079bp)*50ng=25.25 ng @ (10 ng/uL) = 8.41 uL Scaled: 4.57uL"'
  • J04500 /SP + KaiC /XP digested today and yesterday, respectively.
    • BB vector: 50ng @ (5 ng/uL) = 10 uL Scaled: 3.51uL"'
    • BB insert: 3*(~1700/2079bp)*50ng=122.65 ng @ (10 ng/uL) = 18.5 uL Scaled: 6.49 uL"'

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. We will use 1:3 for all constructs; prehaps this works best for all but KaiB, but I figure it doesn't hurt to have more insert / i dont trust the nanodrop.

Add 10uL of #1 to each, then add 1uL of #3. Sit on bench for 30 min, proceed to transformation.

Transformations of J04500\X-P + KaiA/B/C\X-P and J04500\S-P + KaiA/B/C\X-P ligations

We used the full ligation volume (~20 uL) in our transformation, with 25 uL Top10 competent cells.
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