Team:EPF-Lausanne/Notebook/10 August 2012

From 2012.igem.org

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{{:Team:EPF-Lausanne/Template/LabPresence|Diego David Sander Shreya Mouna Alexandra}}
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== Digestion of TNFR + PGL ==
== Digestion of TNFR + PGL ==
; Comments:
; Comments:
-
Growth on all the plates ( about 20 colonies on ost plates and a single colony on the control plate).
+
Growth on all the plates (about 20 colonies on ost plates and a single colony on the control plate).
-
All miniprep colonies except SEAP1 + LS ( there wasn't a lot of growth, we used the 1:2 dilution)
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All miniprep colonies except SEAP1 + LS (there wasn't a lot of growth, we used the 1:2 dilution).
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- 5 µl Buffer N4 10x
- 5 µl Buffer N4 10x
- 5 µl BSA 10x
- 5 µl BSA 10x
-
- 1 µl xbaI ( 20 u / µl )
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- 1 µl XbaI ( 20 u / µl )
- 5 µl DNA
- 5 µl DNA
- 34 µl H20
- 34 µl H20
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-
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; Comments:
; Comments:
 +
Re-verification of excised LovTAP & pGL fragments with a single digestion.
 +
Digestion of pGL backbone with NotI fragments generated 2922 and 1342 bp using small quantities of DNA.
-
Re-verification of excised lovTap & PGL fragments with a single digestion.
+
Quantities in each tube:
-
Digestion of PGL backbone with NOtI fragments generated 2922 and 1342 bp using small quantities of DNA.
+
-
quantities in each tube:
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- 5 µl buffer N2 10x
-
 
+
- 5 µl BSA 10x
-
-5 µl buffer N2 10x
+
- 5 µl DNA ( PGL backbone)
-
-5 µl BSA 10x
+
- 1µl Not1
-
-5 µl DNA ( PGL backbone)
+
- 34 µl H20
-
-1µl Not1
+
-
-34 µl H20
+
------------------------
------------------------
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quantities in each tube:
+
Quantities in each tube:
- 5µl buffer N2 10x
- 5µl buffer N2 10x
- 5µl BSA 10x
- 5µl BSA 10x
- 1µl Xba1
- 1µl Xba1
-
- 5µl DNA (excised LovTap
+
- 5µl DNA (excised LovTAP)
- 34µl H20
- 34µl H20
 +
= Production of a new TAE solution =
= Production of a new TAE solution =
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; Comments:
; Comments:
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+
The TAE in the lab got sticky and contained unknown particles in it, so it was useful to make a new mix.
-
the TAE in the lab got sticky and contained unknown particles in it so it was useful to make a new mix.
+
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Revision as of 00:15, 23 September 2012



Contents

Digestion of TNFR + PGL

Comments

Growth on all the plates (about 20 colonies on ost plates and a single colony on the control plate). All miniprep colonies except SEAP1 + LS (there wasn't a lot of growth, we used the 1:2 dilution).


Protocol used for the Mastermix : - 5 µl Buffer N4 10x - 5 µl BSA 10x - 1 µl XbaI ( 20 u / µl ) - 5 µl DNA - 34 µl H20


Protocol: Restriction site digestion


  1. Look for the best pair of restriction sites, ideally with similar digestion temperatures and times.
    1. [http://tools.neb.com/NEBcutter2/ NEBcutter] for finding cutting enzymes.
    2. [http://www.neb.com/nebecomm/DoubleDigestCalculator.asp Double Digest Finder] for the parameters.
  2. Calculate the amounts required of:
    1. DNA
    2. Buffer (usually from 10x to 1x)
    3. BSA, if needed (usually from 100x to 1x)
    4. Enzymes (depends on the amount of DNA)
    5. Water
  3. Get the recommended buffer (and BSA if needed) from the freezer and let defreeze.
  4. Mix all the ingredients, except DNA, in a tube.
  5. Note: Enzymes should stay no longer than a couple of minutes out of the freezer. Don't touch the bottom of the tubes! Don't vortex!
  6. Distribute the mix in as many tubes as DNA samples and add the DNA.
  7. Keep in the Thermomixer at the recommended temperature.

Sowmya's recommended amounts (50 µl total solution):

  • 5 µl of 10x buffer
  • 0.5 µl of 100x BSA
  • 1 µl of each enzyme
  • 5 µl of DNA
  • 37.5 (up to 50 µl) of water.

Protocol based on what was done on July the 4th.


Comments

Re-verification of excised LovTAP & pGL fragments with a single digestion. Digestion of pGL backbone with NotI fragments generated 2922 and 1342 bp using small quantities of DNA.

Quantities in each tube:

- 5 µl buffer N2 10x - 5 µl BSA 10x - 5 µl DNA ( PGL backbone) - 1µl Not1 - 34 µl H20

Quantities in each tube:

- 5µl buffer N2 10x - 5µl BSA 10x - 1µl Xba1 - 5µl DNA (excised LovTAP) - 34µl H20

Production of a new TAE solution

Protocol: TAE


To make 200 ml of %0x TAE :

  • Tris base: 78.4 g
  • Acetic Acid (100%): 11.4 ml
  • EDTA: 20ml 0.5 M
    • 292.25M --> 146.12 = 0.5 mol --> 1.46 g in 10 ml is 0.5M


Comments

The TAE in the lab got sticky and contained unknown particles in it, so it was useful to make a new mix.

Wiki Links

Important pages

Pages

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