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== MDA Samples Collection from MDA Device v7 (MEF #2)== * Continued from yesterday # Samples were collected by TE buffer after 1.5 hour at 20C. TE buffer: 10mM Tris, 1mM EDTA, 0.2% Tween20 * note: a few valve lines were emptied over the course of 15 hours. Air was pushed into the fluidic layer. # TE buffer was loaded from the top. And the samples were all pushed lower and out of the chambers to avoid mixing. The center 8 chambers were collected first. And the samples of some chambers were back flowed. * Bubbles were generated from empty valve lines during sample collection. Video was taken when bubbles pushed the samples away from chamber 23, and that caused mixing. * note: back flowed can be avoided in the future by designing 2 sets of valves near the collection outlets. # Samples were collected by pipet tips and transferred to PCR tubes. # All chambers were collected in about 6ul volume. # 65C for 15min to deactivate Phi29 == qPCR Recipe and Verification of MDA == * qPCR recipe per sample # 8.2ul water # 0.4ul forward B2 # 0.4ul reverse B2 # 10ul KAPA SYBR Fast (2x) # 1ul sample * There were a total of 24 samples; standards of 1ng/ul, 100pg/ul, 10pg/ul, and 1pg/ul; no template control (1ul water); and negative control (1ul of 1:100 dilution in water). * qPCR result of the 2nd single MEF cell experiments is similar to the 1st experiment. * Out of 24 samples, * A total of 8 samples yielded over 1ng product. * A total of 8 samples yielded 500pg-1ng product. * 3 samples yielded 100pg-500pg and 5 samples yielded less than 100pg. * All 8 samples of low yield (<500pg) were coming from the chambers 18-24 [[File:05242013qPCRa.jpg]] {| {{table}} |chamber|| total (pg) |- |1|| 937.82126 |- |2|| 1660.2249 |- |3|| 1587.6281 |- |4|| 1696.0932 |- |5|| 500.50544 |- |6|| 548.40513 |- |7|| 1025.5768 |- |8|| 2111.1235 |- |9|| 1805.3481 |- |10|| 855.75854 |- |11|| 1264.0812 |- |12|| 715.56902 |- |13|| 755.17198 |- |14|| 762.9601 |- |15|| 453.42019 |- |16|| 649.84148 |- |17|| 1271.5977 |- |18|| 35.038787 |- |19|| 11.20207 |- |20|| 19.804252 |- |21|| 127.08806 |- |22|| 20.274461 |- |23|| 110.39759 |- |24|| 95.640833 |} == CoRE Fragmentation Protocol == * Only chamber 1-16 were used because they had higher DNA concentration * Prepared reagents dilution and kept on ice # 3.3x exo- Klenow buffer (from epicentre): 30ul 10x buffer + 90ul water # diluted UDG: 1ul UDG (5000U/ml) + 128ul water # diluted endonuclease IV: 1ul endoIV (10,000U/ml) + 128ul water # diluted exo- Klenow: 1ul exo- Klenow + 50ul water # diluted dNTP (from epicentre): 1ul 25mM dNTP + 25ul water *In strip PCR tubes, added # 1ul 3.3x exo- Klenow buffer # 1ul diluted UDG # 1ul diluted endonuclease # 1ul sample from MDA device * 37C for 2 hours and 65C for 15min * Then added to PCR tubes # 1ul diluted exo- Klenow # 1ul diluted dNTP * 37C for 1 hour and 75C for 15min == Post-CoRE Library Construction and PCR Verification (accompany to Noi/NOTES/2013-5-22) == * 16 samples from chambers 1-16 were given to Noi * Noi/NOTES/2013-5-22 == MDA Device v7 Preparation for Single PGP1 Cell Experiment (PGP1 #1)== * Device was set up as described in EricChu:LabNotesMDA/2013-5-20 * After air dried the device, it was left on the microscope overnight
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