Team:Harvard/Template:NotebookDataAugust2

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(New miniprep protocol)
 
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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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*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]]

Latest revision as of 22:45, 19 September 2011