Arichard:Notebook/DirectTn5 InTube

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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