Team:Hawaii/Notebook/2008-08-13

From 2008.igem.org

(Difference between revisions)
(→Verification of Transformants)
(→Transformation)
 
(6 intermediate revisions not shown)
Line 4: Line 4:
== Wetlab work ==
== Wetlab work ==
===Verification of Transformants===
===Verification of Transformants===
-
[[Image:081308colonyPCR.jpg|right|thumb|300px|EtBr stained 2% agarose gel ran at 95V for 1 hour. Five microliters of PCR reaction were loaded into each well.]]
+
[[Image:081308colonyPCR.jpg|right|thumb|300px|EtBr stained 2% agarose gel ran at 95V for 100 min. Five microliters of PCR reaction were loaded into each well.]]
:<strong> Grace</strong>
:<strong> Grace</strong>
{| class=wikitable border=1 align=center
{| class=wikitable border=1 align=center
Line 35: Line 35:
|}
|}
:* Colony PCR of transformants and restreaked colonies from yesterday
:* Colony PCR of transformants and restreaked colonies from yesterday
-
::* Ran on 2.5% agarose gel at 95V for 1 min.
+
::* Ran on 2.5% agarose gel at 95V for 100 min.
:* Restreaked:
:* Restreaked:
::* slr1+GFPf colony 5
::* slr1+GFPf colony 5
::* pilA+GFPf colony 4
::* pilA+GFPf colony 4
-
:::* GFPf likely J33207 due to mix up -- see below)
+
:::* GFPf likely J33207 due to mix up -- see below
 +
 
 +
 
 +
===Plasmid Prep===
 +
<strong> Margaret </strong>
 +
:*OriV1-4 and aadA(BB) 4 **the only correct transformants from yesterday's ligation/transformation
 +
 
 +
===Ligation===
 +
 
 +
<strong> Margaret </strong>
 +
 
 +
:*I corrected my math from yesterday's ligation and re-did the ones that did not ligate correctly
 +
 
 +
<strong> Krystle </strong>
 +
 
 +
:*Ligated using quick ligation buffer and quick ligase:
 +
::*gfpf+B0015(digested by MR)
 +
::*gfpf+B0015(digested by GK)
 +
::*gfp+B0015(digested by MR)
 +
::*B0015(digested by MR) <-- as a control
 +
 
 +
===Transformation===
 +
<strong>Margaret</strong>
 +
 
 +
:*omega ligation from yesterday
 +
:*rep+B0030, P1+B0015, aadA(pRL1383a) +B0030 from today's ligation
 +
 
 +
<strong>Krystle</strong>
 +
:*Transformed using DB3.1
 +
:*Ligation products from today (see above)
 +
::Notes: Initial incubation on ice only 3 minutes, final incubation with SOC only 1 hour
 +
 
 +
===Gel From Yesterday's Colony PCR===
 +
<strong> Margaret </strong>
== Drylab Work ==
== Drylab Work ==

Latest revision as of 02:44, 15 August 2008

Projects Events Resources
Sponsors Experiments Milestones Protocols
Notebook (t) Meetings (t)

Things we did today

Wetlab work

Verification of Transformants

EtBr stained 2% agarose gel ran at 95V for 100 min. Five microliters of PCR reaction were loaded into each well.
Grace
Construct Colony forming units
slr1 + GFPf 69 + 1 cluster of colonies
pilA + GFPf 17
nir+rbs + GFP 0
nir+rbs + slr1 0
nir+rbs + pilA 1
plac+rbs + GFP 0
plac+rbs + slr1 0
plac+rbs + pilA 0
  • Colony PCR of transformants and restreaked colonies from yesterday
  • Ran on 2.5% agarose gel at 95V for 100 min.
  • Restreaked:
  • slr1+GFPf colony 5
  • pilA+GFPf colony 4
  • GFPf likely J33207 due to mix up -- see below


Plasmid Prep

Margaret

  • OriV1-4 and aadA(BB) 4 **the only correct transformants from yesterday's ligation/transformation

Ligation

Margaret

  • I corrected my math from yesterday's ligation and re-did the ones that did not ligate correctly

Krystle

  • Ligated using quick ligation buffer and quick ligase:
  • gfpf+B0015(digested by MR)
  • gfpf+B0015(digested by GK)
  • gfp+B0015(digested by MR)
  • B0015(digested by MR) <-- as a control

Transformation

Margaret

  • omega ligation from yesterday
  • rep+B0030, P1+B0015, aadA(pRL1383a) +B0030 from today's ligation

Krystle

  • Transformed using DB3.1
  • Ligation products from today (see above)
Notes: Initial incubation on ice only 3 minutes, final incubation with SOC only 1 hour

Gel From Yesterday's Colony PCR

Margaret

Drylab Work

Sequencing

Grace
  • Checked sequencing results returned from CORE Hawaii
  • Confirmed:
  • B0015
  • B0030
  • B0034 (one bp off)
  • nir
  • slr
  • slr1 = slr2 huh?
  • Other:
  • nir+rbs
  • No rbs present; only nir
  • Huh? We ligated nir INTO rbs
  • I14032+rbs
  • No plac present; only rbs
  • BB-pRL1383a
  • E0040 (GFP) went in successfully, not J33207
  • WTF? Our plasmid preps were/are mislabeled
  • Redo ligation to get blue/white screen?
  • GFP+tt (reverse only)
  • Low quality read; GFP is not present (segment too small)
  • GFPf+tt (reverse only)
  • lacZ fragment present; no tt
  • Huh? We ligated the part INTO tt
  • GFPf+tt and J33207+tt samples switched?
  • J33207+tt
  • Part did not go in; tt only

Discussion

Quote of the Day

History is the only laboratory we have in which to test the consequences of thought. - Étienne Gilson


[http://manoa.hawaii.edu/ Sponsor_UHM.gif][http://manoa.hawaii.edu/ovcrge/ Sponsor_OVCRGE.gif][http://www.ctahr.hawaii.edu Sponsor_CTAHR.gif]


Retrieved from "http://2008.igem.org/Team:Hawaii/Notebook/2008-08-13"