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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...") |
>Andrew (→In-tube optimization of reaction conditions for direct Tn5059 tagmentation on the C1 Open App IFC) |
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(5 intermediate revisions by the same user not shown) | |||
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Objective | ==In-tube optimization of reaction conditions for direct Tn5059 tagmentation on the C1 Open App IFC== | ||
[[File:ifc2tube.xlsx| Print formatted Excel file outlining the experiment]] | |||
===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}} | ||
Line 77: | Line 82: | ||
|- | |- | ||
| Capture site + 1 + 2 + 3 + 4||5||2.233X||1X|||||||||| | | Capture site + 1 + 2 + 3 + 4||5||2.233X||1X|||||||||| | ||
|- | |||
| | |||
|} | |||
====Condition 3, combining Exo- + Tn5 and Exo- + PCR==== | |||
Condition 3 | |||
Objective | |||
*This protocol tests the combination of the Exo- step with Tn5059. | |||
*According to Trina at Illumina (via Brandon), the new Tn5059 does not require DMF, so it could work in the Exo- buffer. | |||
*Furthermore, Brandon has shown that Tn5059 has acceptable activity at 37 C, as that is the reaction temperature he uses. | |||
*The concept is that Exo- will perform second strand synthesis at 37 C, | |||
and that Tn5059 will come in behind to act on double stranded template. | |||
*This protocol also combines the Exo- step with PCR, relying on a 37 C fill-in before the first denaturing cycle | |||
Note | |||
*ALS and NS do not require concentration adjustments, as they are already optimized for succesive addition | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''Tube controls''' | |||
| align="center" style="background:#f0f0f0;"|'''Scaling factor -->''' | |||
| align="center" style="background:#f0f0f0;"|'''111.11''' | |||
| align="center" style="background:#f0f0f0;"|'''Make for -->''' | |||
| align="center" style="background:#f0f0f0;"|'''10''' | |||
|- | |||
| ||Step||Each (uL)||Running (uL)||Adjustment||Reagent | |||
|- | |||
| ||Starting||0.5||0.5||NA||600 pg DNA | |||
|- | |||
| ||Step 1||1||1.5||NA||ALS | |||
|- | |||
| ||Step 2||1||2.5||NA||NS | |||
|- | |||
| ||Step 3||1||3.5||3.500||Exo-/Tn5059 | |||
|- | |||
| ||Step 4||15||18.5||1.233||ProtQ + heatkill | |||
|- | |||
| ||Step 5||15||33.5||2.233||Exo-/PCR | |||
|- | |||
| ||Total||33.5|||||| | |||
|- | |||
| |||||||||| | |||
|- | |||
| Step 1||ALS||1 rxn||10||Adjustment|| | |||
|- | |||
| ||Reagent||Volume (uL)||Volume (uL)||NA|| | |||
|- | |||
| ||H2O||0.75||7.50|||| | |||
|- | |||
| ||5 M KOH --> 400 mM||0.08||0.80|||| | |||
|- | |||
| ||1 M DTT --> 10 mM||0.10||1.00|||| | |||
|- | |||
| ||0.5 M EDTA --> 10 mM||0.02||0.20|||| | |||
|- | |||
| ||20X C1 Loading Reagent||0.05||0.50|||| | |||
|- | |||
| ||Total||1.00||10.00|||| | |||
|- | |||
| |||||||||| | |||
|- | |||
| Step 2||NS||1 rxn||10||Adjustment|| | |||
|- | |||
| ||Reagent||Volume (uL)||Volume (uL)||NA|| | |||
|- | |||
| ||H2O||0.45||4.50|||| | |||
|- | |||
| ||2 M Tris-HCl (pH 7.5) --> 600 mM||0.30||3.00|||| | |||
|- | |||
| ||2 M HCl --> 400 mM||0.20||2.00|||| | |||
|- | |||
| ||20X C1 Loading Reagent||0.05||0.50|||| | |||
|- | |||
| ||Total||1.00||10.00|||| | |||
|- | |||
| |||||||||| | |||
|- | |||
| Step 3||Exo-/Tn5059||1 rxn||10||Adjustment|| | |||
|- | |||
| ||Reagent||Volume (uL)||Volume (uL)||3.500|| | |||
|- | |||
| ||H2O||0.00||0.05|||| | |||
|- | |||
| ||10X Exo- buffer --> 1X||0.35||3.50|||| | |||
|- | |||
| ||25 mM dNTPs --> 400 uM||0.06||0.56|||| | |||
|- | |||
| ||1 mM N6 --> 50 uM||0.18||1.75|||| | |||
|- | |||
| ||10 U/ul Klenow Exo- --> 1 U/ul||0.35||3.50|||| | |||
|- | |||
| ||25X Tn5059 A/B --> 0.1X||0.01||0.14|||| | |||
|- | |||
| ||20X C1 Loading Reagent||0.05||0.50|||| | |||
|- | |||
| ||Total||1.00||10.00|||| | |||
|- | |||
| |||||||||| | |||
|- | |||
| Step 4||ProtQ + heatkill||1 rxn||10||Adjustment|| | |||
|- | |||
| ||Reagent||Volume (uL)||Volume (uL)||1.233|| | |||
|- | |||
| ||H2O||14.07||140.65|||| | |||
|- | |||
| ||20 mg/ml Qiagen Protease --> 0.2 mg/ml||0.18||1.85|||| | |||
|- | |||
| ||20X C1 Loading Reagent||0.75||7.50|||| | |||
|- | |||
| ||Total||15.00||150.00|||| | |||
|- | |||
| |||||||||| | |||
|- | |||
| Step 5||Exo-/PCR||1 rxn||10||Adjustment|| | |||
|- | |||
| ||Reagent||Volume (uL)||Volume (uL)||2.233|| | |||
|- | |||
| ||H2O||4.87||48.71|||| | |||
|- | |||
| ||10X Advantage 2 PCR Buffer --> 1X||3.35||33.50|||| | |||
|- | |||
| ||50X dNTP Mix (Advantage 2) --> 1X||0.67||6.70|||| | |||
|- | |||
| ||10 uM N5xx --> 200 nM||0.67||6.70|||| | |||
|- | |||
| ||10 uM N7xx --> 200 nM||0.67||6.70|||| | |||
|- | |||
| ||50X Advantage 2 Polymerase Mix --> 1X||0.67||6.70|||| | |||
|- | |||
| ||10 U/ul Klenow Exo- --> 1 U/uL||3.3495||33.50|||| | |||
|- | |||
| ||20X C1 Loading Reagent||0.75||7.50|||| | |||
|- | |||
| ||Total||15.00||150.00|||| | |||
|- | |- | ||
| | | | ||
|} | |} |
Latest revision as of 23:46, 9 March 2015
In-tube optimization of reaction conditions for direct Tn5059 tagmentation on the C1 Open App IFC[edit]
Print formatted Excel file outlining the experiment
Motivation[edit]
The C1 Open App IFC contains 5 chambers plus the capture site. This places a constraint on protocol design.
Objective[edit]
Test a number of conditions in PCR tubes with a scaled-up volume and select the best condition based on gel intensity.
Plan[edit]
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[edit]
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 | |||||
Condition 3, combining Exo- + Tn5 and Exo- + PCR[edit]
Condition 3
Objective
- This protocol tests the combination of the Exo- step with Tn5059.
- According to Trina at Illumina (via Brandon), the new Tn5059 does not require DMF, so it could work in the Exo- buffer.
- Furthermore, Brandon has shown that Tn5059 has acceptable activity at 37 C, as that is the reaction temperature he uses.
- The concept is that Exo- will perform second strand synthesis at 37 C,
and that Tn5059 will come in behind to act on double stranded template.
- This protocol also combines the Exo- step with PCR, relying on a 37 C fill-in before the first denaturing cycle
Note
- ALS and NS do not require concentration adjustments, as they are already optimized for succesive addition
' | Tube controls | Scaling factor --> | 111.11 | Make for --> | 10 |
Step | Each (uL) | Running (uL) | Adjustment | Reagent | |
Starting | 0.5 | 0.5 | NA | 600 pg DNA | |
Step 1 | 1 | 1.5 | NA | ALS | |
Step 2 | 1 | 2.5 | NA | NS | |
Step 3 | 1 | 3.5 | 3.500 | Exo-/Tn5059 | |
Step 4 | 15 | 18.5 | 1.233 | ProtQ + heatkill | |
Step 5 | 15 | 33.5 | 2.233 | Exo-/PCR | |
Total | 33.5 | ||||
Step 1 | ALS | 1 rxn | 10 | Adjustment | |
Reagent | Volume (uL) | Volume (uL) | NA | ||
H2O | 0.75 | 7.50 | |||
5 M KOH --> 400 mM | 0.08 | 0.80 | |||
1 M DTT --> 10 mM | 0.10 | 1.00 | |||
0.5 M EDTA --> 10 mM | 0.02 | 0.20 | |||
20X C1 Loading Reagent | 0.05 | 0.50 | |||
Total | 1.00 | 10.00 | |||
Step 2 | NS | 1 rxn | 10 | Adjustment | |
Reagent | Volume (uL) | Volume (uL) | NA | ||
H2O | 0.45 | 4.50 | |||
2 M Tris-HCl (pH 7.5) --> 600 mM | 0.30 | 3.00 | |||
2 M HCl --> 400 mM | 0.20 | 2.00 | |||
20X C1 Loading Reagent | 0.05 | 0.50 | |||
Total | 1.00 | 10.00 | |||
Step 3 | Exo-/Tn5059 | 1 rxn | 10 | Adjustment | |
Reagent | Volume (uL) | Volume (uL) | 3.500 | ||
H2O | 0.00 | 0.05 | |||
10X Exo- buffer --> 1X | 0.35 | 3.50 | |||
25 mM dNTPs --> 400 uM | 0.06 | 0.56 | |||
1 mM N6 --> 50 uM | 0.18 | 1.75 | |||
10 U/ul Klenow Exo- --> 1 U/ul | 0.35 | 3.50 | |||
25X Tn5059 A/B --> 0.1X | 0.01 | 0.14 | |||
20X C1 Loading Reagent | 0.05 | 0.50 | |||
Total | 1.00 | 10.00 | |||
Step 4 | ProtQ + heatkill | 1 rxn | 10 | Adjustment | |
Reagent | Volume (uL) | Volume (uL) | 1.233 | ||
H2O | 14.07 | 140.65 | |||
20 mg/ml Qiagen Protease --> 0.2 mg/ml | 0.18 | 1.85 | |||
20X C1 Loading Reagent | 0.75 | 7.50 | |||
Total | 15.00 | 150.00 | |||
Step 5 | Exo-/PCR | 1 rxn | 10 | Adjustment | |
Reagent | Volume (uL) | Volume (uL) | 2.233 | ||
H2O | 4.87 | 48.71 | |||
10X Advantage 2 PCR Buffer --> 1X | 3.35 | 33.50 | |||
50X dNTP Mix (Advantage 2) --> 1X | 0.67 | 6.70 | |||
10 uM N5xx --> 200 nM | 0.67 | 6.70 | |||
10 uM N7xx --> 200 nM | 0.67 | 6.70 | |||
50X Advantage 2 Polymerase Mix --> 1X | 0.67 | 6.70 | |||
10 U/ul Klenow Exo- --> 1 U/uL | 3.3495 | 33.50 | |||
20X C1 Loading Reagent | 0.75 | 7.50 | |||
Total | 15.00 | 150.00 | |||