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=== qPCR, quantify how much lambda DNA leftover from conversion=== ** First I purified amplified converted lambda DNA using primer set D and quantified the first dilution using Qubit. Got 2.48 ng/uL ** Next, I made serial dilution by 10 folds less each time, using 10 mM Tris-HCl (pH 8.5), .5% Tween 20 solution ** For each sample, I diluted 400X (or 1000X for sample 1), using 199.5 uL 10 mM Tris-HCl (pH 8.5), .5% Tween 20 solution ** Made a master mix then add master mix to triplicates of Standards and samples. {| class="wikitable" | align="center" style="background:#f0f0f0;"|'''Reagent''' | align="center" style="background:#f0f0f0;"|'''Starting concentration''' | align="center" style="background:#f0f0f0;"|'''Volume (uL)''' | align="center" style="background:#f0f0f0;"|'''Final concentration''' | align="center" style="background:#f0f0f0;"|'''50X''' |- | iQ Supermix Green ||2X||5||1X||250 |- | Primer F/R ||10 uM||0.3||300 nM||15 |- | Template||NA||4.7||NA||NA |} * Results from iQ looked ok but after running PAGE gel, amplification were of backgrounds or primer dimers. I need to repeat this using more diluted Standards as well. * qPCR, repeat with Phusion U {| class="wikitable" | align="center" style="background:#f0f0f0;"|'''Reagent''' | align="center" style="background:#f0f0f0;"|'''Starting concentration''' | align="center" style="background:#f0f0f0;"|'''Volume (uL)''' | align="center" style="background:#f0f0f0;"|'''Final concentration''' | align="center" style="background:#f0f0f0;"|'''50X''' |- | Nuclease-free water||NA||4.48||NA||224 |- | Sybr Green||50X||0.32||0.8X||16 |- | Phusion HF Buffer||5X||4||1X||200 |- | dNTPs||10mM||0.4||200 nM||20 |- | Primer F/R ||10uM||0.6||300 nM||30 |- | Phusion U HotStart Pol||2 U/uL||0.2||0.02 U/uL||10 |- | Template||||10||NA||--- |} * Results from Phusion U ** After qPCR I ran a gel to confirm that the correct size products were generated. Expected 267 bp band for LambdaD amplicons. ** [[File:control_dna_v3_qPCR_lambdaD_std_gel.jpg | 300px]] [[File:control_dna_v3_qPCR_lambdaD_post_oxBS_gel.jpg | 300px]] [[File:control_dna_v3_qPCR_post-oxbs_lambdaD_std.jpg | 500px]] [[File:control_dna_v3_qPCR_post-oxbs_lambdaD.jpg | 500px]] * Concentration: ** Normalize concentration: sybr(ng/uL) * 48502 bp/267 bp (Std D) * 400 (dilution factor) ** Dilution factor for #1 is 1000, only used 0.2 uL in 200 uL 1. 32.3 ng/uL; 646 ng 2. 19.7 ng/uL; 394 ng 3. 59.4 ng/uL; 1190 ng 4. 8.14 ng/uL; 163 ng 5. NA 6. 8.42 ng/uL; 168 ng 7. 11.1 ng/uL; 223 ng 8. 7.99 ng/uL; 160 ng
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