User:University of Washington/17 July 2008

From 2008.igem.org

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== Lambda Red Recombination of RP4 (Bryan) ==
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- Attempted colony PCR of IES010, strain carrying a genomic tetR cassete, following a protocol previously validated by graduate advisor.  Ran parallel reactions with primers designed both by myself and Scott.  PCR failed.  Will attempt a control PCR using standard BioBrick primers.  Putative error: dNTPs?
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- Innoculated ON broth culture of BW25141/pKD78.
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== AHL synthesis in yeast (Bryan) ==
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Part C0061 from both 2007 and 2008 distribution have failed to transform, by my hands and by Klavins lab.  Will attempt to rescue the part by PCR.  Obtained template DNA for part C0061 from 2007 distribution courtesy Klavins lab.  Nanodropped at 1.3 ng/ul.
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Grad advisor has advised that high salt content may be interfering with transformation.  Also, microdialysed a portion of the C0061 DNA to reduce salt content, in case we attempt transformation again.
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== RP4 Conjugation ==
== RP4 Conjugation ==
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- PEG, LiAc, TE combination plate buffer was made.
- PEG, LiAc, TE combination plate buffer was made.
 +
- Single stranded carrier DNA was denatured in boiling water for 3 minutes, then placed on ice.
- Single stranded carrier DNA was denatured in boiling water for 3 minutes, then placed on ice.
 +
- 1200 uL of yeast in liquid YEPD media was separated into two Eppendorf tubes. These were centrifuged and pelleted, then the supernatant was decanted.
- 1200 uL of yeast in liquid YEPD media was separated into two Eppendorf tubes. These were centrifuged and pelleted, then the supernatant was decanted.
 +
- 5 uL of carrier DNA was pipetted into each tube of pelleted yeast, then 10 uL of pAC88 and 10 uL of sterile water was pipetted into the experimental and control tubes, respectively.
- 5 uL of carrier DNA was pipetted into each tube of pelleted yeast, then 10 uL of pAC88 and 10 uL of sterile water was pipetted into the experimental and control tubes, respectively.
 +
- The mixture was vortexed then centrifuged for 10 seconds to repellet the cells.
- The mixture was vortexed then centrifuged for 10 seconds to repellet the cells.
 +
- The supernatant was poured off, then 100 uL aliquots of plate buffer was used to resuspend the cells.
- The supernatant was poured off, then 100 uL aliquots of plate buffer was used to resuspend the cells.
 +
- The mixture was vortexed again, then centrigued for another 10 seconds.
- The mixture was vortexed again, then centrigued for another 10 seconds.
 +
- Finally, the supernatant was poured off, and 5 uL of DMOS was added.
- Finally, the supernatant was poured off, and 5 uL of DMOS was added.
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- The two mixtures were plated on two leucine-deficient plates then placed in an incubator at 30 degrees Celsius to grow for the weekend.
- The two mixtures were plated on two leucine-deficient plates then placed in an incubator at 30 degrees Celsius to grow for the weekend.
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 +
==LuxR from AraC and TetR ==
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 +
- Received the sequencing results. None had the mutated part. =[
 +
 +
- Plan: Try QuikChange again with negative and positive control, using Dpn1 from Ingrid, let Dpn1 sit for 2-3 hours instead of 1 hour. Also, will design primer for Elowitz's promoter part to do PCR Amplification.
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Back to [[Team:University_of_Washington/Notebook#Notebook]]
Back to [[Team:University_of_Washington/Notebook#Notebook]]

Latest revision as of 23:19, 22 July 2008

Contents

Lambda Red Recombination of RP4 (Bryan)

- Attempted colony PCR of IES010, strain carrying a genomic tetR cassete, following a protocol previously validated by graduate advisor. Ran parallel reactions with primers designed both by myself and Scott. PCR failed. Will attempt a control PCR using standard BioBrick primers. Putative error: dNTPs?

- Innoculated ON broth culture of BW25141/pKD78.

AHL synthesis in yeast (Bryan)

Part C0061 from both 2007 and 2008 distribution have failed to transform, by my hands and by Klavins lab. Will attempt to rescue the part by PCR. Obtained template DNA for part C0061 from 2007 distribution courtesy Klavins lab. Nanodropped at 1.3 ng/ul.

Grad advisor has advised that high salt content may be interfering with transformation. Also, microdialysed a portion of the C0061 DNA to reduce salt content, in case we attempt transformation again.

RP4 Conjugation

Bac-Bac Conjugation protocol #1
·DH5alpha + pCS26-pac
·DH5alpha:RP4 + pCS26-pac

Yeast Shuttle Plasmid

- PEG, LiAc, TE combination plate buffer was made.

- Single stranded carrier DNA was denatured in boiling water for 3 minutes, then placed on ice.

- 1200 uL of yeast in liquid YEPD media was separated into two Eppendorf tubes. These were centrifuged and pelleted, then the supernatant was decanted.

- 5 uL of carrier DNA was pipetted into each tube of pelleted yeast, then 10 uL of pAC88 and 10 uL of sterile water was pipetted into the experimental and control tubes, respectively.

- The mixture was vortexed then centrifuged for 10 seconds to repellet the cells.

- The supernatant was poured off, then 100 uL aliquots of plate buffer was used to resuspend the cells.

- The mixture was vortexed again, then centrigued for another 10 seconds.

- Finally, the supernatant was poured off, and 5 uL of DMOS was added.

- The two mixtures were plated on two leucine-deficient plates then placed in an incubator at 30 degrees Celsius to grow for the weekend.

LuxR from AraC and TetR

- Received the sequencing results. None had the mutated part. =[

- Plan: Try QuikChange again with negative and positive control, using Dpn1 from Ingrid, let Dpn1 sit for 2-3 hours instead of 1 hour. Also, will design primer for Elowitz's promoter part to do PCR Amplification.


Back to Team:University_of_Washington/Notebook#Notebook

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