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==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. ===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. ====Scaling factors==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Step/chamber''' | align="center" style="background:#f0f0f0;"|'''Condition 1''' | align="center" style="background:#f0f0f0;"|'''Condition 2''' | align="center" style="background:#f0f0f0;"|'''Condition 3''' | align="center" style="background:#f0f0f0;"|'''Condition 4''' | align="center" style="background:#f0f0f0;"|'''Condition 5''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' |- | 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==== 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|||| |- | |}
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