More work...
- I started the day by plating the transformed strains that did grow well. Still, results weren't as expected :(
- 1. rtTh Negative Control using Prefix and Suffix.
- 2. rtTh Positive Control using Prefix and Suffix as primers and plasmid 18 with 7+6+5.
- 3. rtTh with Kanamicin primer and the primer for the mutation of the rbs with DNA dilution of 1/10.
--> Mix 1 for 30 μL <--
-H2O ------------> 8μl
-Buffer 3.3 -----> 6μl
-Mg(OAc)2 -----> 3μl
-dNTP's ----------> 5μl
-Primer 1 ------> 3μl
-Primer 2 ------> 3μl
-DNA ------------> 2μl
--> Mix 2 for 20 μL <-- (this reaction will be added after the hot start)
-H2O ---------------------> 10.5μl
-Buffer 3.3 --------------> 9μl
-rtTh polymerase ------> 0.5μl
- The 30 cycles were programed as follows:
- Initialization step: 94°C for 4:30 min.
- Hot start: Stop to add the second mix 2.
- Denaturation step: 94°C for 45 seg.
- Annealing step: 60°C for 45 seg.
- Extension/elongation step: 72°C for 3:50 min.
- Final elongation: 72°C for 10:00 min.
- Final hold: 4°C for ∞.
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