Arichard:Notebook/DirectTn5 InTube: Difference between revisions
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>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 | ==In-tube optimization of reaction conditions for direct Tn5059 tagmentation on the C1 Open App IFC== | ||
===Motivation=== | |||
The C1 Open App IFC contains 5 chambers plus the capture site. This places a constraint on protocol design. | |||
===Objective=== | |||
Test a number of conditions in PCR tubes with a scaled-up volume and select the best condition based on gel intensity. | Test a number of conditions in PCR tubes with a scaled-up volume and select the best condition based on gel intensity. | ||
Plan | ===Plan=== | ||
I will start with the smallest pipetable volume, 0.5 ul, and scale all other volumes proportionately. | 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. | 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. | Because the C1 IFC adds reagents succesively, each step requires a concentration adjustment. | ||
====Scaling factors==== | |||
{| {{table}} | {| {{table}} |
Revision as of 23:39, 9 March 2015
In-tube optimization of reaction conditions for direct Tn5059 tagmentation on the C1 Open App IFC
Motivation
The C1 Open App IFC contains 5 chambers plus the capture site. This places a constraint on protocol design.
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.
Scaling factors
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 | |||||