Biomod/2013/Harvard/lab: Difference between revisions
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| Line 35: | Line 35: | ||
:purpose: to purify plasmids from our clone colonies | :purpose: to purify plasmids from our clone colonies | ||
We followed [[Biomod/2013/Harvard/protocols#Qiagen_MiniPrep | MiniPrep]] | :We followed [[Biomod/2013/Harvard/protocols#Qiagen_MiniPrep | MiniPrep]] | ||
# transfer cell cultures into centrifuge tubes. Be careful not to drop the tip in. | # transfer cell cultures into centrifuge tubes. Be careful not to drop the tip in. | ||
| Line 54: | Line 54: | ||
# Centrifuge 1 minute. | # Centrifuge 1 minute. | ||
'''III. | '''III. NanoDrop''' | ||
: We followed [[Biomod/2013/Harvard/protocols#NanoDrop | NanoDrop]] | |||
=== Week 2 === | === Week 2 === | ||
Bla | Bla | ||
Revision as of 08:10, 18 June 2013
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HARVARD
BIODESIGN
BIOMOD 2013
-
<html><a href="http://openwetware.org/wiki/Biomod/2013/Harvard"><li>HOME</li></a></html>
<html><a href="http://openwetware.org/wiki/Biomod/2013/Harvard/introduction"><li>INTRODUCTION</li></a></html>
<html><a href="http://openwetware.org/wiki/Biomod/2013/Harvard/design"><li>DESIGN</li></a></html>
<html><a href="http://openwetware.org/wiki/Biomod/2013/Harvard/methods"><li>METHODS</li></a></html>
<html><a href="http://openwetware.org/wiki/Biomod/2013/Harvard/protocols"><li>PROTOCOLS</li></a></html>
<html><a href="http://openwetware.org/wiki/Biomod/2013/Harvard/team"><li>TEAM</li></a></html>
<html><a href="http://openwetware.org/wiki/Biomod/2013/Harvard/lab"><li>LAB NOTEBOOK</li></a></html>
<html><a href="http://openwetware.org/wiki/Biomod/2013/Harvard/results"><li>RESULTS</li></a></html>
<html><a href="http://openwetware.org/wiki/Biomod/2013/Harvard/references"><li>REFERENCES</li></a></html>
Laboratory Notebook
Week 1
6/10/13
General BioMOD Cloning
- purpose: clone for DH5α pDIMC8 BlaCaM His and DH5α pDIMC8 DsbA BlaCaM
- 5 mL LB
- 100µL 20% glucose
- Cm (34 mg/mL working stock), 7.35 µL of this, final concentration 50 µg/mL
- Add cells
- Inoculate overnight
6/11/13
I. Bacterial Solution: LB Agar Plates
- purpose: to make agar plates for 1L LB-agar
- Mix stock
- 960mL DI water (860 mL if using glucose optional)
- 10g of Tryptone
- 10g of NaCl
- 5g yeast extract
- 15g agar
- Autoclave
- liquid (not dry) 30 min. sterilization/20 min drying
- cool to ~50°C in water bath
- Done under flame
- Add glucose - final concentration 20% glucose, if using 20% glucose then 100 mL, optional
- Add antibiotics to the specified final concentration. e.g. Cm: 50 μg/mL. Working stock: 34 mg/mL ⇒ 735 μL per 500 mL
- Pour onto the petri dishes, pre-mark them by type, e.g. : two black lines: Cm, red line: glucose
- Store
II. Experiment II from 6/10/13 continued: Miniprep (QIAGen)
- purpose: to purify plasmids from our clone colonies
- We followed MiniPrep
- transfer cell cultures into centrifuge tubes. Be careful not to drop the tip in.
- Centrifuge for 5 min. at 4000 rpm. N.B. make sure things are balanced.
- Dump the supernatant into old tube. Later add bleach before pouring down sink.
- Resuspend pellets and tansfer to 1.5mL labeled tube with 250μL P1 Buffer.
- 250 μL P2 Buffer (blue color): mix thoroughly.
- Time since step 2 must be less than 4 minutes: add 350μL of N3 Buffer. Mix thoroughly.
- Centrifuge at 13,000 rpm for 10 minutes.
- Apply the supernatant to QIAprep spin column.
- Centrifuge 1 minute. Discard flow-through.
- Wash w/ 750 μL Buffer PE.
- Centrifuge 1 minute. Discard flow-through.
- 1 min additional centrifuge.
- Column into 1.5 microcentrifuge tube.
- Elute with 50 μL water. 5 minute rest.
- Centrifuge 1 minute.
III. NanoDrop
- We followed NanoDrop
Week 2
Bla
