"
Team:University of Lethbridge/Notebook/Protocols
From 2008.igem.org
(Difference between revisions)
m (→Squeeze 'n Freze Gel Extraction) |
Munima.alam (Talk | contribs) m (→xylE PCR (with Phusion pol.)) |
||
| (5 intermediate revisions not shown) | |||
| Line 3: | Line 3: | ||
===PCR protocols=== | ===PCR protocols=== | ||
====CheZ (Taq pol)==== | ====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)==== | ====CheZ (Phusion pol)==== | ||
| Line 25: | Line 38: | ||
4. Hold at 4C | 4. Hold at 4C | ||
| - | ====Riboswitch==== | + | ====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): | Master Mix for 1 reaction (50 uL): | ||
- 10x Buffer: 5 uL | - 10x Buffer: 5 uL | ||
| Line 56: | Line 90: | ||
3. Final extension: 72 C for 10 minutes, then hold at 4 C. | 3. Final extension: 72 C for 10 minutes, then hold at 4 C. | ||
| - | + | ===Other Protocols=== | |
| - | ==== | + | |
| - | + | ||
| - | + | ||
====Squeeze 'n Freze Gel Extraction==== | ====Squeeze 'n Freze Gel Extraction==== | ||
| Line 68: | Line 99: | ||
-Incubate in 3 gel volume 0.3 M NaAc (pH 7.0) at room temperature for 30 minutes. | -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, | + | -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. | -Transfer the solution to the spin column. | ||
| Line 75: | Line 106: | ||
-Freeze the tube in liquid Nitrogen for 1 minute, then spin at full speed for 15 minutes. | -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. | + | -Precipitate the DNA in 95% Ethanol. Remove the supernatant. |
-Wash in 75% Ethanol. Remove as much ethanol as possible. | -Wash in 75% Ethanol. Remove as much ethanol as possible. | ||
| Line 81: | Line 112: | ||
-Centrifige for 5 minutes then, remove the rest of the ethanol. | -Centrifige for 5 minutes then, remove the rest of the ethanol. | ||
| - | -Let pellet | + | -Let pellet air dry for 10 minutes, this allows all ethanol to evaporate off. |
-Resuspend in TE Buffer (pH 8.0), 10 uL. | -Resuspend in TE Buffer (pH 8.0), 10 uL. | ||
Latest revision as of 04:28, 30 October 2008
Back to The University of Lethbridge Main Notebook
Contents |
PCR protocols
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.
Other Protocols
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.
