IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-8-15: Difference between revisions

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| ||4||J04500||19.5 µL||1.75 µL||SpeI & PstI||0.5 µL, 0.5 µL||Buffer 2 (2.5 µL)||0.25 µL ||2 hrs ||2 ||45C
| ||4||J04500||19.5 µL||1.75 µL||SpeI & PstI||0.5 µL, 0.5 µL||Buffer 2 (2.5 µL)||0.25 µL ||2 hrs ||2 ||45C
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|-
| ||5||KaiA||3uL stock + 16.5uL h20||1.75 µL||XbaI & PstI||0.5 µL, 0.5 µL||Buffer 3 (2.5 µL)||0.25 µL ||2 hrs ||1 ||45C
| ||5||KaiA||3uL stock + 16.5uL h20||1.75 µL||XbaI & PstI||0.5 µL, 0.5 µL||Buffer 3 (2.5 µL)||0.25 µL ||6 hrs ||1 ||45C
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|-
| ||6||KaiB||3uL stock + 16.5 µL h20||1.75 µL||XbaI & PstI||0.5 µL, 0.5 µL||Buffer 3 (2.5 µL)||0.25 µL ||2 hrs ||1 ||45C
| ||6||KaiB||3uL stock + 16.5uL h20||1.75 µL||XbaI & PstI||0.5 µL, 0.5 µL||Buffer 3 (2.5 µL)||0.25 µL ||6 hrs ||1 ||45C
|-
|-
| ||7||KaiC||3uL stock + 16.5uL h20||1.75 µL||XbaI & PstI||0.5 µL, 0.5 µL||Buffer 3 (2.5 µL)||0.25 µL ||2 hrs ||1 ||45C
| ||7||KaiC||3uL stock + 16.5uL h20||1.75 µL||XbaI & PstI||0.5 µL, 0.5 µL||Buffer 3 (2.5 µL)||0.25 µL ||6 hrs ||1 ||45C
|-
| ||8||KaiA||3uL stock + 16.5uL h20||1.75 µL||XbaI & PstI||0.5 µL, 0.5 µL||Buffer 3 (2.5 µL)||0.25 µL ||6 hrs ||1 ||45C
|-
| ||9||KaiB||3uL stock + 16.5uL h20||1.75 µL||XbaI & PstI||0.5 µL, 0.5 µL||Buffer 3 (2.5 µL)||0.25 µL ||6 hrs ||1 ||45C
|-
| ||10||KaiC||3uL stock + 16.5uL h20||1.75 µL||XbaI & PstI||0.5 µL, 0.5 µL||Buffer 3 (2.5 µL)||0.25 µL ||6 hrs ||1 ||45C
|-
|-


| Master Mixes||1|| - || - ||15.75 µL||XbaI & PstI||4.5 µL, 4.5 µL||Buffer 3 (22.5 µL)||2.25 µL || - || - ||49.5 µL
| Master Mixes||1|| - || - ||15.75 µL||XbaI & PstI||3 µL, 3 µL||Buffer 3 (15 µL)||1.5 µL || - || - ||  
|-
|-
| ||2|| - || - ||7 µL||SpeI & PstI||2 µL, 2 µL||Buffer 2 (10 µL)||1 µL || - || - ||22 µL
| ||2|| - || - ||7 µL||SpeI & PstI||2 µL, 2 µL||Buffer 2 (10 µL)||1 µL || - || - ||22 µL

Revision as of 00:59, 15 August 2006

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"
 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
Digests 1 J04500 19.5 µL 1.75 µL XbaI & PstI 0.5 µL, 0.5 µL Buffer 3 (2.5 µL) 0.25 µL 2 hrs 1 45C
2 J04500 19.5 µL 1.75 µL SpeI & PstI 0.5 µL, 0.5 µL Buffer 2 (2.5 µL) 0.25 µL 2 hrs 2 45C
3 J04500 19.5 µL 1.75 µL XbaI & PstI 0.5 µL, 0.5 µL Buffer 3 (2.5 µL) 0.25 µL 6 hrs 1 45C
4 J04500 19.5 µL 1.75 µL SpeI & PstI 0.5 µL, 0.5 µL Buffer 2 (2.5 µL) 0.25 µL 2 hrs 2 45C
5 KaiA 3uL stock + 16.5uL h20 1.75 µL XbaI & PstI 0.5 µL, 0.5 µL Buffer 3 (2.5 µL) 0.25 µL 6 hrs 1 45C
6 KaiB 3uL stock + 16.5uL h20 1.75 µL XbaI & PstI 0.5 µL, 0.5 µL Buffer 3 (2.5 µL) 0.25 µL 6 hrs 1 45C
7 KaiC 3uL stock + 16.5uL h20 1.75 µL XbaI & PstI 0.5 µL, 0.5 µL Buffer 3 (2.5 µL) 0.25 µL 6 hrs 1 45C
Master Mixes 1 - - 15.75 µL XbaI & PstI 3 µL, 3 µL Buffer 3 (15 µL) 1.5 µL - -
2 - - 7 µL SpeI & PstI 2 µL, 2 µL Buffer 2 (10 µL) 1 µL - - 22 µL

Pipet 5.5 into ea.

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