Arichard:Notebook/DirectTn5 InTube: Difference between revisions

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==In-tube optimization of reaction conditions for direct Tn5059 tagmentation on the C1 Open App IFC==
==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===
===Motivation===
Line 90: Line 92:
Objective
Objective
*This protocol tests the combination of the Exo- step with Tn5059.
*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,  
*According to Trina at Illumina (via Brandon), the new Tn5059 does not require DMF, so it could work in the Exo- buffer.
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.
*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,  
*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.
and that Tn5059 will come in behind to act on double stranded template.
Line 182: Line 182:
| ||Total||1.00||10.00||||
| ||Total||1.00||10.00||||
|-
|-
|  
| ||||||||||
|}
|-
 
| Step 4||ProtQ + heatkill||1 rxn||10||Adjustment||
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Step 4'''
| align="center" style="background:#f0f0f0;"|'''ProtQ + heatkill'''
| align="center" style="background:#f0f0f0;"|'''1 rxn'''
| align="center" style="background:#f0f0f0;"|'''10'''
| align="center" style="background:#f0f0f0;"|'''Adjustment'''
|-
|-
| ||Reagent||Volume (uL)||Volume (uL)||1.233
| ||Reagent||Volume (uL)||Volume (uL)||1.233||
|-
|-
| ||H2O||14.07||140.65||
| ||H2O||14.07||140.65||||
|-
|-
| ||20 mg/ml Qiagen Protease --> 0.2 mg/ml||0.18||1.85||
| ||20 mg/ml Qiagen Protease --> 0.2 mg/ml||0.18||1.85||||
|-
|-
| ||20X C1 Loading Reagent||0.75||7.50||
| ||20X C1 Loading Reagent||0.75||7.50||||
|-
|-
| ||Total||15.00||150.00||
| ||Total||15.00||150.00||||
|-
|-
| ||||||||
| ||||||||||
|-
|-
| Step 5||Exo-/PCR||1 rxn||10||Adjustment
| Step 5||Exo-/PCR||1 rxn||10||Adjustment||
|-
|-
| ||Reagent||Volume (uL)||Volume (uL)||2.233
| ||Reagent||Volume (uL)||Volume (uL)||2.233||
|-
|-
| ||H2O||4.87||48.71||
| ||H2O||4.87||48.71||||
|-
|-
| ||10X Advantage 2 PCR Buffer --> 1X||3.35||33.50||
| ||10X Advantage 2 PCR Buffer --> 1X||3.35||33.50||||
|-
|-
| ||50X dNTP Mix (Advantage 2) --> 1X||0.67||6.70||
| ||50X dNTP Mix (Advantage 2) --> 1X||0.67||6.70||||
|-
|-
| ||10 uM N5xx --> 200 nM||0.67||6.70||
| ||10 uM N5xx --> 200 nM||0.67||6.70||||
|-
|-
| ||10 uM N7xx --> 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||
| ||50X Advantage 2 Polymerase Mix --> 1X||0.67||6.70||||
|-
|-
| ||10 U/ul Klenow Exo- --> 1 U/uL||3.3495||33.50||
| ||10 U/ul Klenow Exo- --> 1 U/uL||3.3495||33.50||||
|-
|-
| ||20X C1 Loading Reagent||0.75||7.50||
| ||20X C1 Loading Reagent||0.75||7.50||||
|-
|-
| ||Total||15.00||150.00||
| ||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