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

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(Gel purifications of KaiA/B/C\X-P from last night's digest)
(Yet another digest attempt)
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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.
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.
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===Today's Digest Gel===
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[[Image:8-15_Digest_2.jpg |thumb|left|"click for legend"]]
== GFP device issues ==
== GFP device issues ==

Revision as of 16:37, 15 August 2006



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
  • Inoculate KaiABC for a midiprep
  • Run Western Blots

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.

Today's Digest Gel

"click for legend"
"click for legend"

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.
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