Team:University of Lethbridge/Notebook/Protocols

From 2008.igem.org

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(xylE PCR - revise once results are obtained)
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====Competency====
====Competency====
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====Squeeze 'n Freze Gel Extraction====
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-Run the sample you wish to extract on a TAE-Agarose Gel
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-Cut out the band you wish to purify
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-Incubate in 1 gel volume 0.3 M NaCOOH at room temperature for 30 minutes.
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-Make your own spin column from a small microfuge tube with a hole cut out of the bottom,
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stuffed with glass wool. This tube should be inserted inside a 1.5 mL microfuge tube.
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 +
-Transfer the solution to the spin column.
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-Freeze the tube in liquid Nitrogen for 1 minute, then spin at full speed for 15 minutes.
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-Precipitate the DNA in Ethanol. Remove the supernatant.
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-Wash in 75% Ethanol. Remove as much ethanol as possible.
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-Centrifige for 5 minutes then, remove the rest of the ethanol.
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-Let pellet to air dry for 10 minutes, this allows all ethanol to evaporate off.
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-Resuspend in TE Buffer, 10 uL.
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-Quantify either by Gel or UV Spec.
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 +
-Proceed with ligation.

Revision as of 18:35, 11 October 2008

Back to The University of Lethbridge Main Notebook

Contents

PCR protocols

CheZ (Taq pol)

CheZ (Phusion pol)

Conditions for one reaction (25 uL): 

-1x HF buffer (5 uL 5x buffer)
-Forward primer (1.5 uL)
-Reverse primer (1.5 uL)
-dNTPs (0.5 uL)
-Phusion (0.25 uL)
-mQH2O (15.25 uL)
-Template (1 uL)

Cycling protocol ("cheZ"): 

1. Initial denaturation @ 98C for 4 mins (1 cycle)
2a. Denaturation @ 98 C for 30 sec (35 cycles for step #2)
 b. Annealing 47.0C for 30 seconds
 c. Extension @ 72C for 30 sec
3. Final extension @ 72C for 7 mins (1 cycle)
4. Hold at 4C

Riboswitch

rspA TIR

Other protocols

xylE PCR (with Phusion pol.) Master Mix for 1 reaction (50 uL): 

- 10x Buffer: 5 uL
- 10 mM dNTPs: 1 uL
- 50 mM Mg2+: 1.5 uL
- 10 uM RF: 1 uL
- 10 uM RR: 1 uL
- Phusion polymerase: 0.2 uL
- H20: 39.3 uL
- template: 1 uL

Cycle conditions "xylE": 

1. Denaturation: 94 C for 1 min
2. a. Denaturation: 94 C for 30 seconds
   b. Annealing: 52 C for 30 seconds
   c. Extension: 70 C for 30 seconds
   Repeat Step 2 for 30 cycles
3. Final extension: 72 C for 10 minutes, then hold at 4 C.


Competency

Squeeze 'n Freze Gel Extraction

-Run the sample you wish to extract on a TAE-Agarose Gel

-Cut out the band you wish to purify

-Incubate in 1 gel volume 0.3 M NaCOOH at room temperature for 30 minutes.

-Make your own spin column from a small microfuge tube with a hole cut out of the bottom, 
stuffed with glass wool. This tube should be inserted inside a 1.5 mL microfuge tube.

-Transfer the solution to the spin column.

-Freeze the tube in liquid Nitrogen for 1 minute, then spin at full speed for 15 minutes.
 
-Precipitate the DNA in Ethanol. Remove the supernatant.

-Wash in 75% Ethanol. Remove as much ethanol as possible.

-Centrifige for 5 minutes then, remove the rest of the ethanol.

-Let pellet to air dry for 10 minutes, this allows all ethanol to evaporate off.

-Resuspend in TE Buffer, 10 uL.

-Quantify either by Gel or UV Spec.

-Proceed with ligation.
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