EricChu:LabNotesMDA/2013-5-21

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MDA Samples Collection from MDA Device v7 (MEF #2)[edit]

  • Continued from yesterday
  1. 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.
  1. 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.
  1. Samples were collected by pipet tips and transferred to PCR tubes.
  2. All chambers were collected in about 6ul volume.
  3. 65C for 15min to deactivate Phi29

qPCR Recipe and Verification of MDA[edit]

  • qPCR recipe per sample
  1. 8.2ul water
  2. 0.4ul forward B2
  3. 0.4ul reverse B2
  4. 10ul KAPA SYBR Fast (2x)
  5. 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

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[edit]

  • Only chamber 1-16 were used because they had higher DNA concentration
  • Prepared reagents dilution and kept on ice
  1. 3.3x exo- Klenow buffer (from epicentre): 30ul 10x buffer + 90ul water
  2. diluted UDG: 1ul UDG (5000U/ml) + 128ul water
  3. diluted endonuclease IV: 1ul endoIV (10,000U/ml) + 128ul water
  4. diluted exo- Klenow: 1ul exo- Klenow + 50ul water
  5. diluted dNTP (from epicentre): 1ul 25mM dNTP + 25ul water
  • In strip PCR tubes, added
  1. 1ul 3.3x exo- Klenow buffer
  2. 1ul diluted UDG
  3. 1ul diluted endonuclease
  4. 1ul sample from MDA device
  • 37C for 2 hours and 65C for 15min
  • Then added to PCR tubes
  1. 1ul diluted exo- Klenow
  2. 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)[edit]

  • 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)[edit]

  • Device was set up as described in EricChu:LabNotesMDA/2013-5-20
  • After air dried the device, it was left on the microscope overnight