Team:Hawaii/Notebook/2008-08-31

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(Difference between revisions)
(New page: {{Team:Hawaii/Header}} = Things we did today = == Wetlab work == ===Construction of p+r (cont.) and re-replacement of BB-pRL1833a MCS=== :<strong> Grace</strong> [[Image:083108REdigests.j...)
(→Construction of p+r (cont.) and re-replacement of BB-pRL1833a MCS)
 
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[[Image:083108REdigests.jpg|right|thumb|200px|EtBr stained 0.8% agarose gel ran at 60V for 1.5 hours.]]
[[Image:083108REdigests.jpg|right|thumb|200px|EtBr stained 0.8% agarose gel ran at 60V for 1.5 hours.]]
:* Ran RE digests from last night on gel
:* Ran RE digests from last night on gel
 +
::*plac+rbs band not visible; will ligate using plac and rbs parts (instead of plac+rbs part)
 +
:::*Image to right has been edited w/ Photoshop. Faint smear for plac+rbs was not visible before.
:* Extracted bands from gel
:* Extracted bands from gel
:* Ligated:
:* Ligated:
-
::* plac+rbs and C0012 vector
+
::* plac and rbs (B0034) and C0012 vector
::* J33207 and BB-pRL1383a
::* J33207 and BB-pRL1383a
:* Transformed into DH5&alpha; cells
:* Transformed into DH5&alpha; cells
-
::* Plated BB-pRL1383a+J33207 on LB+sp<sub>100</sub>+X-gal and LB+sp<sub>100</sub>+sm<sub>100</sub>+X-gal+IPTG to verify if IPTG is necessary for blue/white screening in this strain
+
::* Plated BB-pRL1383a+J33207 on selective media with and without IPTG to verify if IPTG is necessary for blue/white screening in this strain
 +
 
 +
===Inoculated TB+sp<sub>100</sub> with BB-pRL1383a===
 +
:<strong>Grace</strong>
 +
 
 +
===Construction of Broad-Host-Range Plasmid Parts===
 +
:<strong>Margaret</strong>
 +
 
 +
[[Image:re_digest_8_31_08.jpg|right|thumb|150px|Restriction digest of rep and P1 lytic regions.]]
 +
:*gel of yesterday's re-digest, lane 3 was cleaned using gel purification spin columns and stored in -20&deg;C. Lanes 7-10 were extracted and cleaned as well.
 +
:*Ligation: using T4 ligase, 10X buffer (from the same lot as the enzyme, it was aliquoted today). 2 hour incubation at room temperature. The reaction was placed in 4&deg;C in case transformation does not yield colonies.
 +
 
 +
{|class=wikitable  border=1 align=center
 +
!insert
 +
!vector
 +
|-
 +
|1 pSB1A3 cut with X,P(de-P0<sub>4</sub><sup>2-</sup>)18ng
 +
|3 rep (PCR product) X,P 82ng
 +
|-
 +
|1pSB1A3 cut with X,P(de-P0<sub>4</sub><sup>2-</sup>)35.6ng
 +
|3P1 lytic region X,P 64.4ng
 +
|-
 +
|3pSB1A3 cut with X,P(de-P0<sub>4</sub><sup>2-</sup>)66.2ng
 +
|1 rep (PCR product) X,P 33.8ng\
 +
|}
 +
 
 +
:*Transformation: of ligation products from today into DH5-alpha cells. Everything plated on Amp100 LB plates, pSMC121 used as positive control (plated on SmSp).
 +
 
 +
:* Plasmid Prep: stored in -20&deg;C in 30ul TE buffer
 +
:*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
 +
 
= Discussion =
= Discussion =

Latest revision as of 06:39, 1 September 2008

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

Things we did today

Wetlab work

Construction of p+r (cont.) and re-replacement of BB-pRL1833a MCS

Grace
EtBr stained 0.8% agarose gel ran at 60V for 1.5 hours.
  • Ran RE digests from last night on gel
  • plac+rbs band not visible; will ligate using plac and rbs parts (instead of plac+rbs part)
  • Image to right has been edited w/ Photoshop. Faint smear for plac+rbs was not visible before.
  • Extracted bands from gel
  • Ligated:
  • plac and rbs (B0034) and C0012 vector
  • J33207 and BB-pRL1383a
  • Transformed into DH5α cells
  • Plated BB-pRL1383a+J33207 on selective media with and without IPTG to verify if IPTG is necessary for blue/white screening in this strain

Inoculated TB+sp100 with BB-pRL1383a

Grace

Construction of Broad-Host-Range Plasmid Parts

Margaret
File:Re digest 8 31 08.jpg
Restriction digest of rep and P1 lytic regions.
  • gel of yesterday's re-digest, lane 3 was cleaned using gel purification spin columns and stored in -20°C. Lanes 7-10 were extracted and cleaned as well.
  • Ligation: using T4 ligase, 10X buffer (from the same lot as the enzyme, it was aliquoted today). 2 hour incubation at room temperature. The reaction was placed in 4°C in case transformation does not yield colonies.
insert vector
1 pSB1A3 cut with X,P(de-P042-)18ng 3 rep (PCR product) X,P 82ng
1pSB1A3 cut with X,P(de-P042-)35.6ng 3P1 lytic region X,P 64.4ng
3pSB1A3 cut with X,P(de-P042-)66.2ng 1 rep (PCR product) X,P 33.8ng\
  • Transformation: of ligation products from today into DH5-alpha cells. Everything plated on Amp100 LB plates, pSMC121 used as positive control (plated on SmSp).
  • Plasmid Prep: stored in -20°C in 30ul TE buffer
  • 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

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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