Team:Hawaii/Notebook/2008-08-27

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(Difference between revisions)
(→Construction of p+r)
(margaret)
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::* pSB1A2 (negative control)
::* pSB1A2 (negative control)
:* Transformed DB3.1 cells using 5 μl ligation reaction
:* Transformed DB3.1 cells using 5 μl ligation reaction
 +
 +
===Transformation===
 +
<strong>Margaret</strong>
 +
:* ligation products from yesterday were transformed into DH5-a cells, pSMC121 was used as a positive control
 +
:*rep+pSB1A3 (1:1)
 +
:*rep+pSB1A3 (1:3)
 +
:*rep+pSB1A3 (1:6)
 +
:*P1 lytic +pSB1A3 (1:1)
 +
:*P1 lytic +pSB1A3 (1:3)
 +
:*P1 lytic +pSB1A3 (1:6)
 +
:*[Plac B0030]+pSB1A3 (1:3)
 +
:*[Plac B0030]+pSB1A3 (1:6)
 +
:*[Plac B0034]+pSB1A3 (1:6)**ran out of vector
 +
 +
===Sequencing===
 +
[[Image:sequencing_gel.jpg|right|thumb|150px|Gel of the pcr in preparation for sequencing, also colony pcr of rep and lytic regions.]]
 +
<strong>Margaret</strong>
 +
:*in preparation for sequencing, a PCR was run on the following plasmids:
 +
oriV1-4, aadA(BB)5, aadA(BB)9, aadA(pRL)3, aadA(pRL)6, omega interposon
 +
:*Note: In lane five, it looks like I added oriV instead of aada(BB)5. In the omega lane, there is a ladder. I will still sequence these.
= Discussion =
= Discussion =

Revision as of 03:31, 28 August 2008

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Things we did today

Wetlab work

Construction of p+r

EtBr stained 2% agarose gel ran at 60V for 2.25 hours. Thirty microliters of RE digested DNA were loaded into each well.
Grace
  • Ran RE digested products on gel
  • nir had three bands. Band at ~730bp = ?
  • BB-pRL1383a -- still couldn't see cut out of GFP. Assumed double digest successful, too little DNA to see GFP.
  • J33207 wrong size
  • Extracted nir, C0012 vector, BB-pRL1383a from gel
  • 3A assembly (ligation) of C0012 vector (pSB1A2) with:
  • nir and rbs (B0034)
  • plac and rbs(B0034)
  • pSB1A2 (negative control)
  • Transformed DB3.1 cells using 5 μl ligation reaction

Transformation

Margaret

  • ligation products from yesterday were transformed into DH5-a cells, pSMC121 was used as a positive control
  • rep+pSB1A3 (1:1)
  • rep+pSB1A3 (1:3)
  • rep+pSB1A3 (1:6)
  • P1 lytic +pSB1A3 (1:1)
  • P1 lytic +pSB1A3 (1:3)
  • P1 lytic +pSB1A3 (1:6)
  • [Plac B0030]+pSB1A3 (1:3)
  • [Plac B0030]+pSB1A3 (1:6)
  • [Plac B0034]+pSB1A3 (1:6)**ran out of vector

Sequencing

Gel of the pcr in preparation for sequencing, also colony pcr of rep and lytic regions.

Margaret

  • in preparation for sequencing, a PCR was run on the following plasmids:

oriV1-4, aadA(BB)5, aadA(BB)9, aadA(pRL)3, aadA(pRL)6, omega interposon

  • Note: In lane five, it looks like I added oriV instead of aada(BB)5. In the omega lane, there is a ladder. I will still sequence these.

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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