Team:Hawaii/Notebook/2008-08-30

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(New page: {{Team:Hawaii/Header}} = Things we did today = == Wetlab work == ===Construction of p+r and rereplacement of BB-pRL1383a MCS=== :<strong> Grace</strong> [[Image:083008REdigests.jpg|right|...)
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= Things we did today =
= Things we did today =
== Wetlab work ==
== Wetlab work ==
-
===Construction of p+r and rereplacement of BB-pRL1383a MCS===
+
===Construction of p+r and re-replacement of BB-pRL1383a MCS===
:<strong> Grace</strong>
:<strong> Grace</strong>
[[Image:083008REdigests.jpg|right|thumb|400px|EtBr stained 2% (L) and 0.8% (R) agarose gels ran at 60V for 2 hours and 1 hour, respectively. Thirty microliters of RE digest reaction were loaded into each well.]]
[[Image:083008REdigests.jpg|right|thumb|400px|EtBr stained 2% (L) and 0.8% (R) agarose gels ran at 60V for 2 hours and 1 hour, respectively. Thirty microliters of RE digest reaction were loaded into each well.]]
:* Ran RE digests from last night on agarose gel
:* Ran RE digests from last night on agarose gel
-
:* Extracted bands from gel
+
::* C0012: correct vector band ~2.1kb
 +
::* J33207: band at ~700bp (only one enzyme cut -- XbaI did not cut; PstI cuts because C0012 was cut by PstI) and ~850bp (uncut PCR product)
 +
::* nir: ~800bp band (from GFP contaminant in plasmid prep, cut only once) and ~2kb band (??)
 +
::* BB-pRL1383a: ran WAY too much product. Will redo RE digest with 20% of plasmid used. Since J33207 was only cut once, it's assumed BB-pRL1383a was cut only once as well.
 +
:* Extracted C0012 vector band from gel
:* Ligated:
:* Ligated:
-
::* BB-pRL1383a with J33207
 
-
::* nir + rbs (B0034)
 
::* plac + rbs (B0034)
::* plac + rbs (B0034)
-
:* RE digested p+r with EcoRI, XbaI, SpeI, PstI
+
:* PCR of J33207
 +
:* RE digested:
 +
::* p+r with EcoRI, XbaI, SpeI, PstI
 +
::* J33207 and BB-pRL1383a with EcoRI and PstI
 +
:* Treated C0012 vector (pSB1A2) with SAP
 +
 
 +
===Construction of Broad-host-Range plasmid===
 +
:<strong>Margaret</strong>
 +
[[Image:PCR_8_30_08.jpg|right|thumb|150px|PCR of rep, p1 omega.]]
 +
:*PCR:
 +
::*rep(50ul rxn), pRL1383a (template), rep(primers)
 +
::*P1 lytic(50ul rxn), pSB2K3(extracted from BB paper), P1(primers)
 +
::*(+) control: Base Vector + VF2&VR, (-) control: water + VF2&VR
 +
:*Culture: 5mL Terrific Broth + amp100
 +
::*plac+B0030 (1:6):3, 4, 7
 +
::*plac+B0030 :3
 +
::*plac+B0034 (1:6):1
 +
::*plac+B0034 (1:3):3
 +
 
 +
===PCR===
 +
:<strong>Margaret</strong>
 +
:*P1 lytic (50ul rxn, pSB2K3(extracted from BB paper), P1(primers), 0.1ug/ml BSA
 +
:*omega (25ul rxn) The last gel had a ladder (so does this reaction, see gel from [[Team:Hawaii/Notebook/2008-08-31|tomorrow]]).
 +
 
 +
==Dry Lab==
 +
===Added parts to registry===
 +
:<strong>Margaret</strong>
 +
 
 +
:*plac+B0030:
 +
:*plac+B0034:
 +
:*oriT: K125320
 +
:*P1 lytic: K125330
 +
:*oriV:K125340
 +
:*rep proteins:K125800
 +
:*rep+oriV
= Discussion =
= Discussion =

Latest revision as of 04:14, 1 September 2008

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

Things we did today

Wetlab work

Construction of p+r and re-replacement of BB-pRL1383a MCS

Grace
EtBr stained 2% (L) and 0.8% (R) agarose gels ran at 60V for 2 hours and 1 hour, respectively. Thirty microliters of RE digest reaction were loaded into each well.
  • Ran RE digests from last night on agarose gel
  • C0012: correct vector band ~2.1kb
  • J33207: band at ~700bp (only one enzyme cut -- XbaI did not cut; PstI cuts because C0012 was cut by PstI) and ~850bp (uncut PCR product)
  • nir: ~800bp band (from GFP contaminant in plasmid prep, cut only once) and ~2kb band (??)
  • BB-pRL1383a: ran WAY too much product. Will redo RE digest with 20% of plasmid used. Since J33207 was only cut once, it's assumed BB-pRL1383a was cut only once as well.
  • Extracted C0012 vector band from gel
  • Ligated:
  • plac + rbs (B0034)
  • PCR of J33207
  • RE digested:
  • p+r with EcoRI, XbaI, SpeI, PstI
  • J33207 and BB-pRL1383a with EcoRI and PstI
  • Treated C0012 vector (pSB1A2) with SAP

Construction of Broad-host-Range plasmid

Margaret
File:PCR 8 30 08.jpg
PCR of rep, p1 omega.
  • PCR:
  • rep(50ul rxn), pRL1383a (template), rep(primers)
  • P1 lytic(50ul rxn), pSB2K3(extracted from BB paper), P1(primers)
  • (+) control: Base Vector + VF2&VR, (-) control: water + VF2&VR
  • Culture: 5mL Terrific Broth + amp100
  • plac+B0030 (1:6):3, 4, 7
  • plac+B0030 :3
  • plac+B0034 (1:6):1
  • plac+B0034 (1:3):3

PCR

Margaret
  • P1 lytic (50ul rxn, pSB2K3(extracted from BB paper), P1(primers), 0.1ug/ml BSA
  • omega (25ul rxn) The last gel had a ladder (so does this reaction, see gel from tomorrow).

Dry Lab

Added parts to registry

Margaret
  • plac+B0030:
  • plac+B0034:
  • oriT: K125320
  • P1 lytic: K125330
  • oriV:K125340
  • rep proteins:K125800
  • rep+oriV

Discussion

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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