Team:Hawaii/Notebook/2008-08- 6

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(→Prepared PCR'd nir and J33207 (from yesterday) for transformation/ligation)
(→Sequencing)
 
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== Wetlab work ==
== Wetlab work ==
===Checked transformants from yesterday===
===Checked transformants from yesterday===
 +
[[Image:080608 colony pcr.jpg|right|thumb|200px|EtBr stained 2% agarose gel ran at 95V for 1 hour. Ten microliters of each colony PCR reaction were loaded into each well. Positive control was a nir colony PCR (previously confirmed) and negative control was water.]]
:<strong> Grace</strong>
:<strong> Grace</strong>
-
 
{|class=wikitable border=1 align=center  
{|class=wikitable border=1 align=center  
!Construct
!Construct
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::* Incubated at 37C for 2 hours
::* Incubated at 37C for 2 hours
:* Ran RE digests on EtBr stained 2.0% agarose gel at 72V for 90 min.
:* Ran RE digests on EtBr stained 2.0% agarose gel at 72V for 90 min.
 +
::* No visible bands for nir and J33207 RE digest
 +
:::* We lost a LOT of DNA in the gel purification.
 +
::* Band for J33207 plasmid prep RE digest should be ~650bp
 +
:::* Band at ~850bp = VF2-VR = RE didn't cut???
 +
:* Extracted J33207 band (~850) from gel
 +
:* Ligated 4.5 &mu;l J33207 with 1.5 &mu;l B0015 (tt)
 +
[[Image: 080608nirJ33207pcr.jpg|left|thumb|200px|EtBr stained 2% agarose gel ran at 60V for 100 min. Seven microliters of the PCR reactions were loaded into each lane.]][[Image: 080608nirJ33207pcrcutout.jpg|left|thumb|200px|nir (300bp) and (incorrect) J33207 (1.5kb) bands excised from an EtBr stained 2% agarose gel ran at 60V for 100 min.]][[Image: 080608nirJ33207RE.jpg|left|thumb|200px|EtBr stained 2% agarose gel ran at 72V for 100 min. Fifty microliters of the restriction digests were loaded into each well. ]][[Image: 080608nirJ33207REcut.jpg|left|thumb|200px|J33207 plasmid prep band at ~850bp excised from an EtBr stained 2% agarose gel ran at 72V for 100 min.]]
 +
:<strong>Krystle</strong>
 +
:* Ligated using quick ligase buffer:
 +
:** 3 &mu;l gfpf with 1 &mu;l B0015 (tt)
 +
:** 3 &mu;l gfp with 1 &mu;l B0015 (tt)
 +
:** 3 &mu;l nir with 1 &mu;l B0030 (rbs)
 +
:** 3 &mu;l I14032 with 1 &mu;l B0030 (rbs)
 +
 +
:* Used 5 &mu;l ligation reaction to transform 50 &mu;l DB3.1 cells with the the 5 ligations mentioned above
===Made LB+amp<sub>100</sub> plates===
===Made LB+amp<sub>100</sub> plates===
:<strong> Grace and Margaret</strong>
:<strong> Grace and Margaret</strong>
 +
 +
===Plasmid Prep===
 +
:<strong> Margaret </strong>
 +
 +
:*pSMC121 was plasmid prepped today
 +
 +
:<strong> Krystle </strong>
 +
:*Started 200 ml preps of GFPf, nir, and pRL1383aM
 +
 +
===PCR===
 +
:<strong> Margaret </strong>
 +
[[Image:rep_aada_oriV.jpg|right|thumb|300px|PCR amplification of large quantities of rep, aada, and oriV.]]
 +
:*made large quantities of aadA, rep, and oriV. Please refer to [[Team:Hawaii/PCR Amplification of pRL1383a|The experiment write-up]] for more details.
==Drylab work==
==Drylab work==
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:* Reply from CORE Hawaii
:* Reply from CORE Hawaii
 +
::* They will resequence slr1, slr2, BB-pRL1383a for free due to potential mix up of samples and poor quality reads
 +
::* For samples that were overloaded when 20ng/100bp sequencing guideline used, try 5 ng/100bp instead.
 +
= Discussion =
= Discussion =
 +
:* We're low on small nitrile gloves and Qiagen spin columns. Need to order more. '''NW'''
 +
:* Be careful when pouring agar plates. Bubbles suck!
-
= Quote of the Day =
 
-
<blockquote>''History is the only laboratory we have in which to test the consequences of thought.'' - Étienne Gilson</blockquote>
 
{{Team:Hawaii/Footer}}
{{Team:Hawaii/Footer}}
__NOTOC__
__NOTOC__

Latest revision as of 16:41, 9 August 2008

Projects Events Resources
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Notebook (t) Meetings (t)

Things we did today

Wetlab work

Checked transformants from yesterday

EtBr stained 2% agarose gel ran at 95V for 1 hour. Ten microliters of each colony PCR reaction were loaded into each well. Positive control was a nir colony PCR (previously confirmed) and negative control was water.
Grace
Construct Colony forming units
I14032 (plac) + B0030 (rbs) 3
pnir + B003 (rbs) 0
E0040 (GFP) + B0015 (tt) 1
GFPf + B0015 (tt) 2 + 2 clusters of colonies
  • Colony PCR'd transformants
  • 30 cycles, anneal at 62C, extend for 1 min.
  • Ran on EtBr stained 2.0% agarose gel at 95V for 1 hour
  • None of the transformations were successful :o(

Determined DNA concentrations of purified RE'd PCR products from 8/4

Grace
DNA sample Concentration
nir 17.0 ng/μl
slr1 31.3 ng/μl
slr2 11.8 ng/μl
pilA 13.0 ng/μl
GFP 12.0 ng/μl
GFPf 12.2 ng/μl
E0240 8.6 ng/μl
I14032 16.8 ng/μl

Prepared PCR'd nir and J33207 (from yesterday) for transformation/ligation

Grace
  • Ran PCR products on EtBr stained 2.0% agarose gel at 60V for 100 min.
  • nir band at 330bp confirmed
  • J33207 = 4 bands (1.5kb, 2.5kb, 3.2kb, 10kb), none of which are correct
  • [http://www.partsregistry.org partsregistry] says J33207 DNA is inconsistent
  • Extracted nir and 1.5kb J33207 band from gel
  • RE digested in 50 μl rxns with EcoRI and SpeI in NEBuffer 2
  • Larger rxn volume may improve digest efficiency
  • Incubated at 37C for 2.5 hours
  • RE digested 10 μl J33207 plasmid prep with EcoRI and SpeI in NEBuffer 2
  • To confirm plasmid prep; if good, will use for ligation rxn
  • Incubated at 37C for 2 hours
  • Ran RE digests on EtBr stained 2.0% agarose gel at 72V for 90 min.
  • No visible bands for nir and J33207 RE digest
  • We lost a LOT of DNA in the gel purification.
  • Band for J33207 plasmid prep RE digest should be ~650bp
  • Band at ~850bp = VF2-VR = RE didn't cut???
  • Extracted J33207 band (~850) from gel
  • Ligated 4.5 μl J33207 with 1.5 μl B0015 (tt)
EtBr stained 2% agarose gel ran at 60V for 100 min. Seven microliters of the PCR reactions were loaded into each lane.
nir (300bp) and (incorrect) J33207 (1.5kb) bands excised from an EtBr stained 2% agarose gel ran at 60V for 100 min.
EtBr stained 2% agarose gel ran at 72V for 100 min. Fifty microliters of the restriction digests were loaded into each well.
J33207 plasmid prep band at ~850bp excised from an EtBr stained 2% agarose gel ran at 72V for 100 min.
Krystle
  • Ligated using quick ligase buffer:
    • 3 μl gfpf with 1 μl B0015 (tt)
    • 3 μl gfp with 1 μl B0015 (tt)
    • 3 μl nir with 1 μl B0030 (rbs)
    • 3 μl I14032 with 1 μl B0030 (rbs)
  • Used 5 μl ligation reaction to transform 50 μl DB3.1 cells with the the 5 ligations mentioned above

Made LB+amp100 plates

Grace and Margaret

Plasmid Prep

Margaret
  • pSMC121 was plasmid prepped today
Krystle
  • Started 200 ml preps of GFPf, nir, and pRL1383aM

PCR

Margaret
PCR amplification of large quantities of rep, aada, and oriV.

Drylab work

Sequencing

Grace
  • Reply from CORE Hawaii
  • They will resequence slr1, slr2, BB-pRL1383a for free due to potential mix up of samples and poor quality reads
  • For samples that were overloaded when 20ng/100bp sequencing guideline used, try 5 ng/100bp instead.

Discussion

  • We're low on small nitrile gloves and Qiagen spin columns. Need to order more. NW
  • Be careful when pouring agar plates. Bubbles suck!


[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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