Team:University of Lethbridge/Notebook/Protocols

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CheZ (Taq pol)
From September 28, 2008 in the "Chemotaxis" lab book: - Tempate DNA 1uL - 10X Econo Taq Buffer 2.5uL - dNTP mix 1uL - Primer 1 5uL - Primer 2 5uL - Econo Taq 0.25uL - ddH2O 19.75uL

Cycling Conditions: - Incubate PCR Reactions 2 min at 94 C - Denature 30 sec at 94 C  - Anneal 30 sec at 47 C  - Extend 1 min/kb at 72 C  - Final extension 7 min at 72 C  - Hold indefinitely at 4 C

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 (Taq pol)
From October 14, 2008 in the "Riboswitch" notebook: Objective: PCR of the riboswitch in a 50 uL x 9 reactions.

Master Mix (1x): -10x Buffer: 5 uL -10 mM dNTP: 1 uL - 50 mM MgCl2: 1.5 uL - 10 mM reverse primer: 1 uL -10 mM forward primer: 1 uL -Taq polymerase: 0.2 uL -d2H2O: 39.3 uL

rspA TIR (Taq pol)
From September 30, 2008 in "rpsa TIR" notebook

Reaction Conditions: - 1.0 uL Template DNA - 5.0 uL 10x Buffer - 2.0 uL 10 mM dNTPs - 1.0 uL Forward Primer - 1.0 uL Reverse Primer - 0.5 uL Econo taq polymerase - 39.5 uL Optima H2O

Cycling Conditions: 1. 94 C 2 min 2. a. 94 C 15 sec b. 47 C 15 sec c. 72 C 15 sec 3. Repeat step 2 for 25 cycles 4.72 C 5 min

xylE (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.

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 3 gel volume 0.3 M NaAc (pH 7.0) at room temperature for 30 minutes. -Make your own spin column from a small microfuge tube with a hole cut out of the bottom, involves flamage and a wire, stuff 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 95% 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 air dry for 10 minutes, this allows all ethanol to evaporate off. -Resuspend in TE Buffer (pH 8.0), 10 uL. -Quantify either by Gel or UV Spec. -Proceed with ligation.