Team:Hawaii/Notebook/2008-08-11

From 2008.igem.org

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:::* Genomic DNA up top?
:::* Genomic DNA up top?
:::* Clean prep (no RNA)!
:::* Clean prep (no RNA)!
-
:::* Only E0240 verified. All other bands wrong size (circular/supercoiled?)
+
:::* Only E0240 verified. All other bands wrong size (circular/supercoiled?). Need RE digest to verify.
:* Checked DNA concentrations via nanodrop spectrometer
:* Checked DNA concentrations via nanodrop spectrometer
{|class=wikitable border=1 align=center
{|class=wikitable border=1 align=center
Line 44: Line 44:
|}
|}
-
===Made 1000x Amp,sub>100</sub> stock solution===
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===Made 1000x Amp<sub>100</sub> stock solution===
:<strong>Grace</strong>
:<strong>Grace</strong>
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!DNA concentration
!DNA concentration
|-
|-
-
|nir+rbs
+
|align=center|nir+rbs
-
|4.8 ng/&mu;l
+
|align=center|4.8 ng/&mu;l
|-
|-
-
|plac+rbs
+
|align=center|plac+rbs
-
|3.6 ng/&mu;l
+
|align=center|3.6 ng/&mu;l
|-
|-
-
|GFP
+
|align=center|GFP
-
|4.7 ng/&mu;l
+
|align=center|4.7 ng/&mu;l
|-
|-
-
|GFPfusion
+
|align=center|GFPfusion
-
|6.4 ng/&mu;l
+
|align=center|6.4 ng/&mu;l
|}
|}
-
:* Restriction digested in 30 &mu;l reactions:
+
[[Image:081108REdigests.jpg|right|thumb|200px|EtBr stained 2% agarose gel ran at 72V for 1.5 hours. Thirty microliters of the RE digest reactions were loaded into each well.]]
 +
:* Restriction digested in 30 &mu;l reactions, incubated at 37C for 2 hours:
::* B0015 with XbaI then EcoRI
::* B0015 with XbaI then EcoRI
::* GFP and GFPf with EcoRI and SpeI
::* GFP and GFPf with EcoRI and SpeI
::* slr1, slr2, pilA with SpeI and PstI
::* slr1, slr2, pilA with SpeI and PstI
-
:* Ran new RE digests EtBr stained 2% agarose gel at 72V for 2 hours
+
:* Ran new RE digests EtBr stained 2% agarose gel at 72V for 1.5 hours
:* Extracted parts from gel and determined DNA concentrations
:* Extracted parts from gel and determined DNA concentrations
 +
{|class=wikitable border=1 align=center
 +
!Part
 +
!DNA concentration
 +
|-
 +
|align=center|slr1
 +
|align=center|2.6 ng/&mu;l
 +
|-
 +
|align=center|pilA
 +
|align=center| 1.1 ng/&mu;l
 +
|-
 +
|align=center|GFP
 +
|align=center|0.4 ng/&mu;l
 +
|-
 +
|align=center|GFPf
 +
|align=center|11.3 ng/&mu;l
 +
|-
 +
|align=center|B0015
 +
|align=center|1.9 ng/&mu;l
 +
|}
 +
:* Ligated for 1 hour using Quick T4 DNA Ligase and Quick Ligase buffer:
 +
::* 8 &mu;l GFP + 0.5 &mu;l B0015
 +
::* 4 &mu;l GFPf + 4 &mu;l B0015
 +
::* 2 &mu;l GFPf + 1.5 &mu;l slr1
 +
::* 2 &mu;l GFPf + 3.5 &mu;l pilA
 +
:* Transformed 7 &mu;l ligation reaction into DB3.1 cells
 +
:* RE digest overnight of 22 &mu;l pSB1A2 with EcoRI and PstI for 3A assembly
 +
 +
===Testing restriction enzymes in the lab's -20C freezer===
 +
:<strong>Grace</strong>
 +
 +
:* Digested pRL1383a with BamHI (should result in a single linear fragment)
 +
:* Digested pRL1383a with HindIII (should result in a single linear fragment)
 +
:* Digested plasmid preps (E0240, I14032, I51020, nir+rbs, plac+rbs) with NotI (should result in two fragments -- vector and insert)
 +
 +
===[[Team:Hawaii/Ligation of pRL1383a Parts|Ligation of pRL1383a Parts]]===
 +
[[Image:re_digest_8_11_08.jpg|right|thumb|200px|Restriction digest after 2 hours.]]
 +
[[Image:Ligation_rxn_8_11_08.jpg|right|thumb|200px|Ligation reaction after 2 hours.]]
 +
:<strong> Margaret </strong>
 +
 +
:*restriction digest of rep, oriV, aada(BB), aada(pRL1383a), P1 lytic region, pSB1A3, B0030, B0015
 +
:*Ligation: rep+B0030, oriV+pSB1A3, aadA(BB)+B0030, aadA(pRL1383a)+B0030, P1 lytic + B0015, pSB1A3 to itself (-) control
 +
:*Transformation into DH5-a (batch 3)
 +
 +
===Started Culture for plasmid prep & cryostocks===
 +
:* to be completed 8/12
 +
:*B0015, pSB3K3, oriT(cryostock & plasmid prep), B0030, I14032, E0040, J33207
= Discussion =
= Discussion =
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= Quote of the Day =
= Quote of the Day =
<blockquote>''History is the only laboratory we have in which to test the consequences of thought.'' - Étienne Gilson</blockquote>
<blockquote>''History is the only laboratory we have in which to test the consequences of thought.'' - Étienne Gilson</blockquote>
-
 
+
<br><br><br><br><br><br><br><br><br><br><br>
{{Team:Hawaii/Footer}}
{{Team:Hawaii/Footer}}
__NOTOC__
__NOTOC__

Latest revision as of 06:46, 12 August 2008

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

Things we did today

Wetlab work

Checked plasmid prep from weekend

Grace
EtBr stained 0.8% agarose gel ran at 95V for 1 hour. Five microliters of plasmid were loaded into each well.
  • Ran on 2.0% agarose gel to verify plasmids
  • DNA didn't run. Agarose concentration too high. Redid on 0.8% gel.
  • Genomic DNA up top?
  • Clean prep (no RNA)!
  • Only E0240 verified. All other bands wrong size (circular/supercoiled?). Need RE digest to verify.
  • Checked DNA concentrations via nanodrop spectrometer
Plasmid DNA concentration 260/280 260/230
E0240 757.7 ng/μl 2.06 1.49
I14032 (2005 distribution) 541.4 ng/μl 2.01 1.27
I51020 2775.6 ng/μl 1.97 1.77
nir+rbs 566.8 ng/μl 1.83 1.10
plac+rbs 344.0 ng/μl 1.95 1.28

Made 1000x Amp100 stock solution

Grace

Reinoculated for cryostocking

Grace
  • I14032 from 2005 and 2008 distributions

Construction of GFP device

Grace
  • Extracted nir+rbs, plac+rbs, GFP, GFPf from gel ran yesterday
  • B0015 could not be extracted because fragment was not visible under short wave UV
  • Digestion was done for 3A assembly rather than rear ligation (oops). Redid RE digest.
  • Checked DNA concentrations via nanodrop spectrometer
Part DNA concentration
nir+rbs 4.8 ng/μl
plac+rbs 3.6 ng/μl
GFP 4.7 ng/μl
GFPfusion 6.4 ng/μl
EtBr stained 2% agarose gel ran at 72V for 1.5 hours. Thirty microliters of the RE digest reactions were loaded into each well.
  • Restriction digested in 30 μl reactions, incubated at 37C for 2 hours:
  • B0015 with XbaI then EcoRI
  • GFP and GFPf with EcoRI and SpeI
  • slr1, slr2, pilA with SpeI and PstI
  • Ran new RE digests EtBr stained 2% agarose gel at 72V for 1.5 hours
  • Extracted parts from gel and determined DNA concentrations
Part DNA concentration
slr1 2.6 ng/μl
pilA 1.1 ng/μl
GFP 0.4 ng/μl
GFPf 11.3 ng/μl
B0015 1.9 ng/μl
  • Ligated for 1 hour using Quick T4 DNA Ligase and Quick Ligase buffer:
  • 8 μl GFP + 0.5 μl B0015
  • 4 μl GFPf + 4 μl B0015
  • 2 μl GFPf + 1.5 μl slr1
  • 2 μl GFPf + 3.5 μl pilA
  • Transformed 7 μl ligation reaction into DB3.1 cells
  • RE digest overnight of 22 μl pSB1A2 with EcoRI and PstI for 3A assembly

Testing restriction enzymes in the lab's -20C freezer

Grace
  • Digested pRL1383a with BamHI (should result in a single linear fragment)
  • Digested pRL1383a with HindIII (should result in a single linear fragment)
  • Digested plasmid preps (E0240, I14032, I51020, nir+rbs, plac+rbs) with NotI (should result in two fragments -- vector and insert)

Ligation of pRL1383a Parts

Restriction digest after 2 hours.
Ligation reaction after 2 hours.
Margaret
  • restriction digest of rep, oriV, aada(BB), aada(pRL1383a), P1 lytic region, pSB1A3, B0030, B0015
  • Ligation: rep+B0030, oriV+pSB1A3, aadA(BB)+B0030, aadA(pRL1383a)+B0030, P1 lytic + B0015, pSB1A3 to itself (-) control
  • Transformation into DH5-a (batch 3)

Started Culture for plasmid prep & cryostocks

  • to be completed 8/12
  • B0015, pSB3K3, oriT(cryostock & plasmid prep), B0030, I14032, E0040, J33207

Discussion

  • FYI:
  • According to the Endy lab, ligation reactions should have <100ng DNA per reaction for maximum efficiency
  • ~10ng vector should be used in ligation reactions (6:1 ratio of insert to vector)

Quote of the Day

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












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