Talk:Team:IvyTech-South Bend/19 October 2010

From 2010.igem.org

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(→B-Galactosidase Activity Assay -- Marian Price-Carter, 9/7/00)
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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
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

Revision as of 18:31, 27 October 2010


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

  1. 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

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