Difference between revisions of "Dandekar & Chandler:Screen for Bactobolin Resistant Mutants"

From OpenWetWare
Jump to: navigation, search
(New page: Back home: Dandekar & Chandler:Protocols ==Screen for Bactobolin Resistant Mutants== '''Day 1:''' 1. Streak WT ''B. subtilis'' onto LB and incubate at 37C. :a. JH642: Transformable ...)
Line 1: Line 1:
Back home: [[Dandekar & Chandler:Protocols]]
<div style="padding: 10px; color: #ffffff; background-color: #39275B; margin: width: 100% ">
[[Dandekar & Chandler | <font face="trebuchet ms" style="color:#C79900"> '''Home''' </font>]] &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
[[Dandekar & Chandler:Contact | <font face="trebuchet ms" style="color:#C79900"> '''Contact''' </font>]] &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
[[Dandekar & Chandler:Back Door | <font face="trebuchet ms" style="color:#C79900"> '''Internal''' </font>]] &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
[[Dandekar & Chandler:Protocols | <font face="trebuchet ms" style="color:#C79900"> '''Protocols''' </font>]] &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
[[Dandekar & Chandler:Lab Members | <font face="trebuchet ms" style="color:#C79900"> '''Lab Members''' </font>]] &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
[[Dandekar & Chandler:Publications | <font face="trebuchet ms" style="color:#C79900"> '''Publications''' </font>]] &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
[[Dandekar & Chandler:Research | <font face="trebuchet ms" style="color:#C79900"> '''Research''' </font>]] &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
[[Dandekar & Chandler:Talks | <font face="trebuchet ms" style="color:#C79900"> '''Talks''' </font>]] &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
==Screen for Bactobolin Resistant Mutants==
==Screen for Bactobolin Resistant Mutants==

Latest revision as of 13:48, 11 October 2013

Home        Contact        Internal        Protocols        Lab Members        Publications        Research        Talks       

Screen for Bactobolin Resistant Mutants

Day 1:

1. Streak WT B. subtilis onto LB and incubate at 37C.

a. JH642: Transformable strain
b. 3610: Wild strain that was able to mutate L2

2. Streak WT B. thailandensis onto LB and incubate at 37C.

Day 2:

1. Inoculate one colony into a tube with 5 mL LB.

2. Inoculate ~5 colonies of B. thailandensis into each of 3 baffled flasks with 10 mL LB.

3. Incubate all o/n at 30C shaking.

Day 3:

1. Harvest B. thailandensis supernatant:

a. Pellet cells in microcentrifuge and keep supernatant
b. Filter/sterilize supernatant twice with 0.22 uM filters
c. Store supernatant at 4C

2. Pour LB plates supplemented with various amounts of supernatant. Aim for a concentration where growth is just inhibited.

a. 100-150 uL supernatant per 20 mL LB agar worked best for this screen.

3. Spread 100 uL undiluted B. subtilis culture onto each plate.

4. Incubate at 30C for 1-2 days.

Day 4 or 5:

1. Restreak potential resistant isolates onto fresh supernatant plate alongside WT as a negative control. Incubate at 30C for 1-2 days.