Daniel:Notebook/ComboLock/2017-3-6: Difference between revisions
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After reading some papers I'm going to give it one more go to see if I can't amplify the DNA from [[Daniel:Notebook/ComboLock/2017-3-3|last week]] better. I'll use the method of largest amplification I know: most time using random hexamers as primers. | After reading some papers I'm going to give it one more go to see if I can't amplify the DNA from [[Daniel:Notebook/ComboLock/2017-3-3|last week]] better. I'll use the method of largest amplification I know: most time using random hexamers as primers. | ||
== | ==RCA Repeat (Maximum Power!)== | ||
<ol> | <ol> | ||
Line 69: | Line 69: | ||
<li>Heat kill enzyme by incubating 10 minutes at 65C; Hold at 12C</li> | <li>Heat kill enzyme by incubating 10 minutes at 65C; Hold at 12C</li> | ||
</ol></ol> | </ol></ol> | ||
Continued [[Daniel:Notebook/ComboLock/2017-3-7|tomorrow]] | |||
[[Category:ComboLock]] [[Category:20170301]] | [[Category:ComboLock]] [[Category:20170301]] |
Latest revision as of 00:18, 7 March 2017
Padlock0601 Test (Started Wed March 1)[edit]
After reading some papers I'm going to give it one more go to see if I can't amplify the DNA from last week better. I'll use the method of largest amplification I know: most time using random hexamers as primers.
RCA Repeat (Maximum Power!)[edit]
- Maximal Rolling Circle Amplification
- Create master mix according to following table
- Add 16 uL master mix to each tube
- Add 4 uL template according to table
- Incubate at 95 C for 5 minutes
- Lower to 55C for 15 minutes
- Lower to 30C for at least 2 minutes
- Add 1 uL Phi29 polymerase to each sample
- Incubate 15 hours at 30C
- Heat kill enzyme by incubating 10 minutes at 65C; Hold at 12C
Reagent | Single Rxn Vol | 16.2X MM uL |
Template | 4 | 0 |
RCA Hexamer (1 mM) | 0.5 | 8.1 |
dNTP (1 mM) | 5 | 81 |
10X Buffer | 2 | 32.4 |
Phi29 | 1 | 0 |
BSA (10 mg/mL) | 0.4 | 6.48 |
nfH2O | 7.1 | 115.02 |
Total | 20 | 243 |
Continued tomorrow