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Team:University of Lethbridge/Notebook/Protocols
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===Other protocols=== | ===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==== | ====Competency==== | ||
Revision as of 00:31, 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.
