Team:Hawaii/Notebook/2008-10- 9

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(New page: {{Team:Hawaii/Header}} = Things we did today = == Wetlab work == ===Construction of secretion device (cont.)=== :<strong> Grace</strong> [[Image:100908REdigest.jpg|right|thumb|200px|EtBr ...)
(→Triparental conjugtion)
 
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:* BBpRL #1, 18 cultures did not grow
:* BBpRL #1, 18 cultures did not grow
:* PCR of colonies (on Xgal plate) showed no insert present (?!?)
:* PCR of colonies (on Xgal plate) showed no insert present (?!?)
-
:* PCR of colonies from non-Xgal plate)...
+
:* PCR of colonies from non-Xgal plate) showed no insert present (?!?)
 +
::* Faint band for colonies #1, 13 at 400bp ???
 +
:* Restreaked original colonies #1, 3, 13, 18 on freshly made LB+sp<sub>100</sub>+sm<sub>100</sub>
 +
::* Original colonies scraped w/ loop. Hopefully there's something there.
 +
 
 +
===Made 5%LB+BG-11 and BG-11+sp<sub>2.5</sub>+sm<sub>2.5</sub> plates===
 +
:<strong>Grace and Krystle</strong>
 +
 
 +
===Construction of plasmid===
 +
:<strong>margaret</strong>
 +
[[Image:rep_aadA_purification_10_9.jpg|right|thumb|300px|plac/rbs/rep5 (E,S), plac/rbs/rep7 (E,S), plac/rbs/aada (ap32 E,S), plac/rbs/aada ap2 cut with X,P.]]
 +
:* Gel purification of re-digest
 +
::*plac/rbs/aada (ap32 was cut with E,S), ap2 did not cut correctly
 +
::*plac/rbs/rep (7 was cut with E,S), rep5 did not cut correctly
 +
:*Ligation, transformation (in DH5 alpha):
 +
::*oriT + plac/rbs/aada + pSB1A3 (dephosphorylated)
 +
::*oriV + plac/rbs/rep + pSB1A3 (dephosphorylated)
 +
::pSB1A3 (dephosphorylated) + self
 +
::*(+) control for transformation- pUC18
 +
::*(-) control for transformation- no plasmid
 +
 
 +
===OriV back-ups===
 +
:<strong>margaret</strong>
 +
[[Image:oriv_verification_10_9.jpg|right|thumb|300px|Verification of oriV transformation.]]
 +
:* two colonies were of correct size from yesterday's transformation, so i did a plasmid prep of them and I will store them in -20 C in TE
= Discussion =
= Discussion =

Latest revision as of 23:44, 11 October 2008

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Things we did today

Wetlab work

Construction of secretion device (cont.)

Grace
File:100908REdigest.jpg
EtBr stained 4% agarose gel ran at 60V for 3.5 hours. Twenty microliters of RE digested product were loaded into each well.
  • Ran RE digests on gel
  • Extracted bands from gel
  • Ligated:
  • J33207 and pRL1383a
  • nir and rbs+GFPf+tt #1 and pSB1A3
  • plac and rbs+GFPf+tt #1 and pSB1A3
  • nir+rbs and slr1+GFPf and pSB1A3
  • nir+rbs and pilA+GFPf+tt and pSB1A3
  • Used 5 μl ligation reaction to transform DH5α

Sequencing

Grace
  • Prepared and sent samples in for sequencing:
  • pilA+GFPf+tt #21 (KS)
  • nir+rbs+slr1+GFPf #15 (10/2 transformation)
  • slr1+GFPf+tt (10/2 transformation)
  • nir+rbs+slr1+GFPf #6 (10/8 transformation)
  • plac+rbs+pilA+GFPf+tt #5, 7, 11 (10/8 transformation)

Triparental conjugtion

Grace
  • BBpRL #1, 18 cultures did not grow
  • PCR of colonies (on Xgal plate) showed no insert present (?!?)
  • PCR of colonies from non-Xgal plate) showed no insert present (?!?)
  • Faint band for colonies #1, 13 at 400bp ???
  • Restreaked original colonies #1, 3, 13, 18 on freshly made LB+sp100+sm100
  • Original colonies scraped w/ loop. Hopefully there's something there.

Made 5%LB+BG-11 and BG-11+sp2.5+sm2.5 plates

Grace and Krystle

Construction of plasmid

margaret
File:Rep aadA purification 10 9.jpg
plac/rbs/rep5 (E,S), plac/rbs/rep7 (E,S), plac/rbs/aada (ap32 E,S), plac/rbs/aada ap2 cut with X,P.
  • Gel purification of re-digest
  • plac/rbs/aada (ap32 was cut with E,S), ap2 did not cut correctly
  • plac/rbs/rep (7 was cut with E,S), rep5 did not cut correctly
  • Ligation, transformation (in DH5 alpha):
  • oriT + plac/rbs/aada + pSB1A3 (dephosphorylated)
  • oriV + plac/rbs/rep + pSB1A3 (dephosphorylated)
pSB1A3 (dephosphorylated) + self
  • (+) control for transformation- pUC18
  • (-) control for transformation- no plasmid

OriV back-ups

margaret
File:Oriv verification 10 9.jpg
Verification of oriV transformation.
  • two colonies were of correct size from yesterday's transformation, so i did a plasmid prep of them and I will store them in -20 C in TE

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]


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