Andrew:Notebook/directTn5150301
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In-tube optimization of reaction conditions for direct Tn5059 tagmentation on the C1 Open App IFC[edit]
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.
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 |
Capture site | 1 | 1.5X | 1X |
Capture site | 1 + 2 | 1.25X | 1X |
Capture site | 1 + 2 + 3 | 1.167X | 1X |
Capture site | 1 + 2 + 3 + 4 | 1.028X = 1X | 1X |
Capture site | 1 + 2 + 3 + 4 + 5 | 1.015X = 1X | 1X |
Capture site + 1 | 2 | 2.5X | 1X |
Capture site + 1 | 2 + 3 | 1.75X | 1X |
Capture site + 1 | 2 + 3 + 4 | 1.088X | 1X |
Capture site + 1 | 2 + 3 + 4 + 5 | 1.047X = 1X | 1X |
Capture site + 1 + 2 | 3 | 3.5X | 1X |
Capture site + 1 + 2 | 3 + 4 | 1.156X | 1X |
Capture site + 1 + 2 | 3 + 4 + 5 | 1.08X | 1X |
Capture site + 1 + 2 + 3 | 4 | 1.233X | 1X |
Capture site + 1 + 2 + 3 | 4 + 5 | 1.117X | 1X |
Capture site + 1 + 2 + 3 + 4 | 5 | 2.233X | 1X |
C1 Open App IFC | ' | ' | ' |
Chamber | Each (nL) | Running (nL) | Adjustment |
C | 4.5 | 4.5 | |
E1 | 9 | 13.5 | 1.5 |
E2 | 9 | 22.5 | 2.5 |
E3 | 9 | 31.5 | 3.5 |
E4 | 135 | 166.5 | 1.233 |
E5 | 135 | 301.5 | 2.233 |
Total | 301.5 | ||
Tube controls | Scaling factor --> | 111.11 | ' |
Step | Each (uL) | Running (uL) | Adjustment |
Starting | 0.5 | 0.5 | |
Step 1 | 1 | 1.5 | 1.5 |
Step 2 | 1 | 2.5 | 2.5 |
Step 3 | 1 | 3.5 | 3.5 |
Step 4 | 15 | 18.5 | 1.233 |
Step 5 | 15 | 33.5 | 2.233 |
Total | 33.5 | ||
Condition 3[edit]
I decided to test condition 3 first.
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
Setup[edit]
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 | ||