EricChu:LabNotesMDA/2013-5-21
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MDA Samples Collection from MDA Device v7 (MEF #2)[edit]
- 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[edit]
- 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
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
- 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)[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