Arichard:Notebook/DirectTn5 InTube

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Revision as of 23:38, 9 March 2015 by >Andrew (Created page with "Objective Test a number of conditions in PCR tubes with a scaled-up volume and select the best condition based on gel intensity. Plan I will start with the smallest pipetable...")
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Objective Test a number of conditions in PCR tubes with a scaled-up volume and select the best condition based on gel intensity.

Plan I will start with the smallest pipetable volume, 0.5 ul, and scale all other volumes proportionately. The scaling factor is 111 = 0.5 ul/4.5 nl, the actual starting volume of the C1 IFC. Because the C1 IFC adds reagents succesively, each step requires a concentration adjustment.



Step/chamber Condition 1 Condition 2 Condition 3 Condition 4 Condition 5 ' ' '
Step 1 ALS ALS ALS ALS HLS/ProtQ + heatkill
Step 2 NS NS NS NS Tn5059
Step 3 Exo- Exo- Exo-/Tn5059 Exo- ProtQ + heatkill
Step 4 Tn5059 Tn5059 ProtQ + heatkill Tn5059 + heatkill PCR
Step 5 ProtQ + heatkill ProtQ + heatkill PCR PCR
Step 6 (Tube only) Exo- + heatkill PCR
Step 7 (Tube only) PCR
Chamber(s) already filled Going into Chamber(s) Added reagents at C1 Loading Reagent C1 Open App IFC
Capture site 1 1.5X 1X Chamber Each (nL) Running (nL) Adjustment
Capture site 1 + 2 1.25X 1X C 4.5 4.5
Capture site 1 + 2 + 3 1.167X 1X E1 9 13.5 1.5
Capture site 1 + 2 + 3 + 4 1.028X = 1X 1X E2 9 22.5 2.5
Capture site 1 + 2 + 3 + 4 + 5 1.015X = 1X 1X E3 9 31.5 3.5
E4 135 166.5 1.233
Capture site + 1 2 2.5X 1X E5 135 301.5 2.233
Capture site + 1 2 + 3 1.75X 1X Total 301.5
Capture site + 1 2 + 3 + 4 1.088X 1X
Capture site + 1 2 + 3 + 4 + 5 1.047X = 1X 1X Tube controls Scaling factor --> 111.11
Step Each (uL) Running (uL) Adjustment
Capture site + 1 + 2 3 3.5X 1X Starting 0.5 0.5
Capture site + 1 + 2 3 + 4 1.156X 1X Step 1 1 1.5 1.5
Capture site + 1 + 2 3 + 4 + 5 1.08X 1X Step 2 1 2.5 2.5
Step 3 1 3.5 3.5
Capture site + 1 + 2 + 3 4 1.233X 1X Step 4 15 18.5 1.233
Capture site + 1 + 2 + 3 4 + 5 1.117X 1X Step 5 15 33.5 2.233
Total 33.5
Capture site + 1 + 2 + 3 + 4 5 2.233X 1X