Team:Hawaii/Notebook/2008-08-31

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(Difference between revisions)
(→Construction of p+r (cont.) and re-replacement of BB-pRL1833a MCS)
(margaret)
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:* Transformed into DH5α cells
:* 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
::* Plated BB-pRL1383a+J33207 on selective media with and without IPTG to verify if IPTG is necessary for blue/white screening in this strain
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===Construction of Broad-Host-Range Plasmid Parts===
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<strong>Margaret</strong>
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[[Image:re_digest_8_31_08.jpg|right|thumb|150px|Restriction digest of rep and P1 lytic regions.]]
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:*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.
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:*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.
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{|class=wikitable  border=1 align=center
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!insert
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!vector
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|-
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|1 pSB1A3 cut with X,P(de-P0<sub>4</sub><sup>2-</sup>)18ng
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|3 rep (PCR product) X,P 82ng
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|-
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|1pSB1A3 cut with X,P(de-P0<sub>4</sub><sup>2-</sup>)35.6ng
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|3P1 lytic region X,P 64.4ng
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|-
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|3pSB1A3 cut with X,P(de-P0<sub>4</sub><sup>2-</sup>)66.2ng
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|1 rep (PCR product) X,P 33.8ng\
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|}
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:*Transformation: of ligation products from today into DH5-alpha cells. Everything plated on Amp100 LB plates, pSMC121 used as positive control (plated on SmSp).
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= Discussion =
= Discussion =

Revision as of 03:24, 1 September 2008

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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)
  • 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

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).



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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