Daniel:Notebook/ComboLock/2017-3-29: Difference between revisions

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==Bglii Digestion==
==Bglii Digestion==


Testing bglii digestion of RCA product. No qPCR to start with (RCA ''should'' be enough)
Testing bglii digestion of RCA product. No qPCR to start with (RCA ''should'' be enough). For recall, here is the sample matrix again and the RCA conditions used.
 
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|- style="background-color:#CCFF66;font-size:12pt;font-weight:bold" align="center"
| width="105" height="30" | Sample
| width="105" | Master Mix
| width="100" | Enzyme
| width="100" | Incubation Time (hrs)
 
|- style="font-size:12pt"
| height="15"  valign="bottom" | X (A-F)
| align="center" valign="bottom" | X
| align="center" valign="bottom" | NEB
| align="center" align="center" valign="bottom" | 3
 
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | Y (A-F)
| align="center" valign="bottom" | Y
| align="center" valign="bottom" | Thermo
| align="center" align="center" valign="bottom" | 3
 
|- style="font-size:12pt"
| height="15"  valign="bottom" | Z (A-F)
| align="center" valign="bottom" | Z
| align="center" valign="bottom" | Epi (1:10)
| align="center" align="center" valign="bottom" | 3
 
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | P (A-F)
| align="center" valign="bottom" | X
| align="center" valign="bottom" | NEB
| align="center" align="center" valign="bottom" | 6
 
|- style="font-size:12pt"
| height="15"  valign="bottom" | Q (A-F)
| align="center" valign="bottom" | X
| align="center" valign="bottom" | NEB
| align="center" align="center" valign="bottom" | 12
 
|}


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Revision as of 17:43, 29 March 2017

RCA Test (Started March 24)

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Bglii Digestion

Testing bglii digestion of RCA product. No qPCR to start with (RCA should be enough). For recall, here is the sample matrix again and the RCA conditions used.

Sample Master Mix Enzyme Incubation Time (hrs)
X (A-F) X NEB 3
Y (A-F) Y Thermo 3
Z (A-F) Z Epi (1:10) 3
P (A-F) X NEB 6
Q (A-F) X NEB 12
  1. Bglii digestion
    1. Make the following reactions; Do not add Bglii yet
    2. Reagent Single Rxn Vol Master Mix (30.2X)
      RCA Rxn 2 0
      Bglii Cutter Primer + (100 uM) 2 60.4
      10X Buffer 3.1 2 60.4
      Bglii 1 0
      nfH2O 13 392.6
      Total 20 513.4
    3. Use the following thermocycler program
      1. 5 min 95C
      2. Ramp to 50C at 0.2C/s
      3. 10 min 50C
      4. 2 hr 37C; When this cycle starts add 1 uL Bglii
      5. Heat kill with 65C for 20 min