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Talk:Team:IvyTech-South Bend/19 October 2010
From 2010.igem.org
Contents |
10/19/10
Today we are doing large scale DNA extractions from E-Coli Recovering t9002 and lac-z
first taking the OD of each tube
after Blacking spectrometer I will run a sample of each tube.
E-coli w/T9002 from 10/15/10 has an OD of .355 @600um
E-Coli w/LacZ from 10/15/10 has an OD of 3.49 @ 600um
Streaked E-coli w/T9002 from 10/12/10 has an OD of .363 @600um
- 2 Streaked E-Coli w/LacZ from 10/15/10 has an OD of 3.26 @ 600um
after I took 4 bottles from spec to hood and removed 5ml from each tube placing into 50 cc tubes with LB/Amp and then placed them int 37C inculbator
Then placed the 4 tubes extracted from and placed them into centrefuge to spin down to extract DNA.
Then following protocol from pg 19. After extracting the DNA I placed into fridge in back Room @ 4C.
Then made LB Broth in 3 1Lt flasks weighing 10 g in each LB mix then adding 500 ml to it, placed into microwave one time on beverage setting then into autoclave on liquid setting 121 C @ sw
jhull
10/19/10
I followed the protocol below
I changed my pipette tip each time to prevent cross contamination
IGEM Protocols/Restriction Digest
From partsregistry.org At iGEM HQ we use this protocol for restriction digests along with enzymes purchased from NEB.
Materials
- PCR tube
- dH20
- Enzymes (EcoRI, XbaI, SpeI, PstI)
- BSA
- Enzyme Buffer (NEBuffer 2)*
Notes: You should keep all materials on ice.
Protocol
1. Add 500n~ of DNA to be digested, and adjust with dH20 for a total volume of 42.5ul.
2. Add 5ul ofNEBuffer 2 to the tube.
3. Add 0.5ul of BSA to the robe.
4. Add l ul of your first enzyme
5. Add l ul ofyour second e~e.(~¢,
6. There should be a total volume of 5(iul. Mix well and spin down.
7. Incubate the restriction digest at_3_7C for 30miri, and then 80C for 20min to heat kill the enzymes. l~e incubate in a thermocycler with a heat~-d lid
8. Run a portion of the digest on a gel, to check that both plasmid and part length are accurate. You may also use 2u of the digest (20mg of DNA) for Ligations.
New restriction and Ligation
Joe and I perfomed new restriction and ligation
We did the sam protocol for the first restriction and ligation
The DNA was put into thermocycler 30 min @ 37C and 20 min @ 80C
Stored @ 4.0C in thermocycler
- unsure of the date
- will be used for electrophoresis
dgarvey
