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Revision as of 21:07, 8 July 2010 (view source) Dspakowicz (Talk | contribs) (New page: ==July 8==) ← Older edit		Latest revision as of 21:24, 8 July 2010 (view source) Dspakowicz (Talk | contribs) (→July 8)
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	==July 8==		==July 8==
		+
		+	The phsABC plasmid worked!  We have metal precipitation!  Woooo!
		+
		+	1. Cloning issues
		+	*Did the double digestion of the terminator work?
		+	** The EcoRI was old and might have failed, check each enzyme on a gel
		+	*SpeI not very efficient in EcoRI buffer, will try to incubate longer
		+	* The ligation didn't work, but there are lots of ways to keep trying.  This is a great way for people to come help during the week.
		+
		+
		+	2. Copper precipitation assay
		+	*Pick a concentration from the cell growth assay, like 1 mM Cu?
		+	*Grow shaking, at time points spin out the cells and measure the absorbance
		+	**this is assaying for loss of copper
		+
		+
		+	3. Does phsABC confer copper resistance?
		+	*96-well plate assay
		+	*same as before, but with the phsABC plasmid
		+	**Since Leah has this assay dialed-in, this is a great job for a part-time helper to run and analyze!
		+
		+	4.  Cu resistance
		+	* will be shipping in a day or two
		+	**pRJ1004 (Cu efflux protein) (Jon will email to follow up)
		+	***massive plasmid, no antibiotic resistance
		+	**Get together next week to request several plasmids (Tuesday morning at 10 am).
		+
		+	5. Email Kara about the mold (Jon)
		+
		+	6. Ways to get students involved (Dan email the group)
		+	*Ask when they're available, delegate a task
		+	*make sure someone is there to help them
		+
		+	==July 15==

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Latest revision as of 21:24, 8 July 2010

July 8

The phsABC plasmid worked! We have metal precipitation! Woooo!

1. Cloning issues

• Did the double digestion of the terminator work?
  • The EcoRI was old and might have failed, check each enzyme on a gel
• SpeI not very efficient in EcoRI buffer, will try to incubate longer
• The ligation didn't work, but there are lots of ways to keep trying. This is a great way for people to come help during the week.

2. Copper precipitation assay

• Pick a concentration from the cell growth assay, like 1 mM Cu?
• Grow shaking, at time points spin out the cells and measure the absorbance
  • this is assaying for loss of copper

3. Does phsABC confer copper resistance?

• 96-well plate assay
• same as before, but with the phsABC plasmid
  • Since Leah has this assay dialed-in, this is a great job for a part-time helper to run and analyze!

4. Cu resistance

• will be shipping in a day or two
  • pRJ1004 (Cu efflux protein) (Jon will email to follow up)
    • massive plasmid, no antibiotic resistance
  • Get together next week to request several plasmids (Tuesday morning at 10 am).

5. Email Kara about the mold (Jon)

6. Ways to get students involved (Dan email the group)

• Ask when they're available, delegate a task
• make sure someone is there to help them

July 15