Team:Harvard/Template:NotebookDataAugust2

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====Glycerol stocks and final platings====
====Glycerol stocks and final platings====
We made glycerol stocks of the final four plasmids in Top 10 ChemComp bacteria, which were Pos Con 77 (85.2), OZ052 (52.1), OZ123 (123.4), and Zif268 (Zif268.1).  We also made final plates of these bacteria, using 1/1000 ul on spec plates.</div>
We made glycerol stocks of the final four plasmids in Top 10 ChemComp bacteria, which were Pos Con 77 (85.2), OZ052 (52.1), OZ123 (123.4), and Zif268 (Zif268.1).  We also made final plates of these bacteria, using 1/1000 ul on spec plates.</div>
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==August 6th==
==August 6th==
===Team ZF===
===Team ZF===
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{|
{|
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  |[[File:HARVHARV2011.8.6_manual_miniprep_cb_bottom_awesome.jpg|thumb|Over 20x times normal yield compared with the Qiagen miniprep kit.]]
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  |[[File:HARV2011.8.6_manual_miniprep_cb_bottom_awesome.jpg|thumb|Over 20x times normal yield compared with the Qiagen miniprep kit.]]
  |}
  |}
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*HisB bands at about 900bp; PyrF bands at about 400bp.
*HisB bands at about 900bp; PyrF bands at about 400bp.
*Results: none of the colonies have both genes knocked out but some do look like they may have the HisB deletion.  We may try again using MAGE round 5 colonies.
*Results: none of the colonies have both genes knocked out but some do look like they may have the HisB deletion.  We may try again using MAGE round 5 colonies.
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[[File: HARV2011.08.06.hispyrKOtest1(labeled).png|thumb|left|HisB and PyrF allele specific PCRs part 1. Yellow-labeled colonies may have a knock out. 8/6/11]]
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[[File:HARV2011.08.06.hispyrKOtest1(labeled)-0016.png|thumb|left|HisB and PyrF allele specific PCRs part 1. Yellow-labeled colonies may have a knock out. 8/6/11]]
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[[File: HARV2011.08.06.pyrfKOtest(labeled).png|thumb|none|Separate E gel of the pyrF PCRs cut off in gel 1. 8/6/11]]
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[[File:HARV2011.08.06.pyrfKOtest(labeled)-0018.png|thumb|none|Separate E gel of the pyrF PCRs cut off in gel 1. 8/6/11]]
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[[File: HARV2011.08.06.hispyrKOtest2_2(labeled).png|thumb|none| HisB and PyrF allele specific PCRs part 2 8/6/11]]
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[[File:HARV2011.08.06.hispyrKOtest2_2(labeled)-0017.png|thumb|none| HisB and PyrF allele specific PCRs part 2 8/6/11]]
'''pSB4K5 digestion:'''
'''pSB4K5 digestion:'''
*Since our minipreps have been giving such low yields, we tried an [http://openwetware.org/wiki/Knight:Miniprep_low_copy_plasmids altered protocol].  The result, however was the same: 6mL of culture produced 19ng/uL (in 30uL)
*Since our minipreps have been giving such low yields, we tried an [http://openwetware.org/wiki/Knight:Miniprep_low_copy_plasmids altered protocol].  The result, however was the same: 6mL of culture produced 19ng/uL (in 30uL)
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**3 hour incubation at 37C
**3 hour incubation at 37C
*gel extraction: the gel was empty. No clue why. Obviously this is a setback--we will have to redo the miniprep and digestion before we can form the whole plasmid.</div>
*gel extraction: the gel was empty. No clue why. Obviously this is a setback--we will have to redo the miniprep and digestion before we can form the whole plasmid.</div>
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==August 7th==
==August 7th==
===Team ZF===
===Team ZF===
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Although the bands are faint, the cut band is somewhere around the correct size (around 2.8 kb).  Although the band appears to be anywhere between 3 kb and 4 kb in the gel, the ladder ran strangely for some reason.  However, there is still a fairly well-defined band instead of a smear in the cut lane.  This in itself is a huge relief - if there were significant genomic DNA contamination, we might expect to get a smear from many differently sized cuts.  Furthermore, the uncut band looks standard for a plasmid, similar to the picture on the website here: [[http://faculty.plattsburgh.edu/donald.slish/Electrophoresis.html]].  There are three bands in our gel as they appear on the website.  However, our linearized plasmid is actually fairly close to the bottom band for the uncut plasmid, which is presumably the supercoiled form.</div>
Although the bands are faint, the cut band is somewhere around the correct size (around 2.8 kb).  Although the band appears to be anywhere between 3 kb and 4 kb in the gel, the ladder ran strangely for some reason.  However, there is still a fairly well-defined band instead of a smear in the cut lane.  This in itself is a huge relief - if there were significant genomic DNA contamination, we might expect to get a smear from many differently sized cuts.  Furthermore, the uncut band looks standard for a plasmid, similar to the picture on the website here: [[http://faculty.plattsburgh.edu/donald.slish/Electrophoresis.html]].  There are three bands in our gel as they appear on the website.  However, our linearized plasmid is actually fairly close to the bottom band for the uncut plasmid, which is presumably the supercoiled form.</div>
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==August 8th==
==August 8th==
===Team Wolfe===
===Team Wolfe===
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*64 degrees annealing and 90 seconds elongation
*64 degrees annealing and 90 seconds elongation
*Gel shows PCR was successful
*Gel shows PCR was successful
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[[File: HARV2011.08.08 His Pyr MAGE for Seq (labeled).png|thumb|none|MAGE sequencing PCR]]
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[[File: HARV2011.08.08 His Pyr MAGE for Seq (labeled)-0019.png|thumb|none|MAGE sequencing PCR]]
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  |[[File:HARV2011.8.TolCKanpresence(labeled).png|thumb|blah]]
  |[[File:HARV2011.8.TolCKanpresence(labeled).png|thumb|blah]]
  |}</div>
  |}</div>
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==August 9th==
==August 9th==
===Team Wolfe===
===Team Wolfe===

Latest revision as of 22:45, 19 September 2011