Revision as of 17:47, 24 April 2015 by >Andrew
In-tube optimization of reaction conditions for direct Tn5059 tagmentation on the C1 Open App IFC[edit]
Experiment spreadsheet
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
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 |
|
|
Results[edit]