Daniel:Notebook/HiResChrPaint/2013-8-8: Difference between revisions

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===qPCR Results===
===qPCR Results===
[[Image:2013-8-8-S2-ULS.png|600px]]


===Nanodrop Results===
===Nanodrop Results===
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  1. Prepare Samples
  1. Prepare Samples


{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="font-size:12pt;font-weight:bold" align="center"
| width="65" height="15" | Sample
| width="65" | V4S2A
| width="65" | V4S2B
| width="65" | V4S2C
| width="65" | V4S2D
| width="65" | V6S2A
| width="65" | V6S2B
| width="65" | V6S2C
| width="65" | V6S2D


|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | ng/uL
| align="center" | 64.7
| align="center" | 62.6
| align="center" | 69.1
| align="center" | 79.2
| align="center" | 139
| align="center" | 126.8
| align="center" | 127.3
| align="center" | 127.2
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | ug in 35 uL
| align="center" | 2.3
| align="center" | 2.2
| align="center" | 2.4
| align="center" | 2.8
| align="center" | 4.9
| align="center" | 4.4
| align="center" | 4.5
| align="center" | 4.5
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="30" | uL 10X buffer
| align="center" | 5
| align="center" | 5
| align="center" | 5
| align="center" | 5
| align="center" | 5
| align="center" | 5
| align="center" | 5
| align="center" | 5
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="30" | uL DpnII (50U/uL)
| align="center" | 2
| align="center" | 2
| align="center" | 2
| align="center" | 2
| align="center" | 2
| align="center" | 2
| align="center" | 2
| align="center" | 2
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | uL nfH2O
| align="center" | 8
| align="center" | 8
| align="center" | 8
| align="center" | 8
| align="center" | 8
| align="center" | 8
| align="center" | 8
| align="center" | 8
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | Total
| align="center" | 50
| align="center" | 50
| align="center" | 50
| align="center" | 50
| align="center" | 50
| align="center" | 50
| align="center" | 50
| align="center" | 50
|}


  2. Incubate samples at 37C for 2 hours; 20 minute heat shock at 65C
  2. Incubate samples at 37C for 16 hours; 20 minute heat shock at 65C; [[Daniel:Notebook/HiResChrPaint/2013-8-9|Continued 8/9/2013]]
3. [[Daniel:Protocols/Qiaquick_Column|Column purification]], elute with 30 uL EB
4. Measure in Nanodrop

Latest revision as of 17:00, 9 August 2013

S2 Probe Prep (ULS)[edit]

Back to Calendar

I have some more Alexa 488 from the ULYSIS kits left, and they recommend using it within a month. Since it has been almost a month since I opened the bottle, I guess I should make more (once coupled the dye is stable for several more months). Besides, I could use the ULS labeled probe as a comparison for future FISH experiments.

qPCR Amplification[edit]

1. Set up master reactions according to table
  20 nM OligoPool 2X Kapa SYBR qPCR MM 100 uM AP1V41U 100 uM AP2V4 100 uM AP1V61U 100 uM AP2V6 50 mM aa-dUTP H20 Total Volume
per rxn 0.2 50 0.4 0.4 0.4 0.4 0 48.2 100
V4S2 (12.5x) 2.5 625 5 5 0 0 0 612.5 1250
V6S2 (12.5x) 2.5 625 0 0 5 5 0 612.5 1250
2. Aliquot 100 uL into each of 12 qPCR tubes (24 in total for both reactions)
3. PCR protocol:
    i. PCR 3 min 95C
    ii. 95C 3 sec
    iii. 55C 30 sec 
    iv. 72C 20 sec
    v. plate read
    vi. goto ii
    vii. 72C 2 min
    viii. 16C hold
4. Pool 3 samples together each (300 uL each, 4 pools per set, 8 total)
5. Column puficiation, elute with 40 uL EB
6. Measure in Nanodrop

qPCR Results[edit]

File:2013-8-8-S2-ULS.png

Nanodrop Results[edit]

Sample V4S2A V4S2B V4S2C V4S2D V6S2A V6S2B V6S2C V6S2D
ng/uL 64.7 62.6 69.1 79.2 139 126.8 127.3 127.2
ug in 40 uL 2.6 2.5 2.8 3.2 5.6 5.1 5.1 5.1
Total ug       11.0       20.8

DpnII Digestion[edit]

1. Prepare Samples
Sample V4S2A V4S2B V4S2C V4S2D V6S2A V6S2B V6S2C V6S2D
ng/uL 64.7 62.6 69.1 79.2 139 126.8 127.3 127.2
ug in 35 uL 2.3 2.2 2.4 2.8 4.9 4.4 4.5 4.5
uL 10X buffer 5 5 5 5 5 5 5 5
uL DpnII (50U/uL) 2 2 2 2 2 2 2 2
uL nfH2O 8 8 8 8 8 8 8 8
Total 50 50 50 50 50 50 50 50
2. Incubate samples at 37C for 16 hours; 20 minute heat shock at 65C; Continued 8/9/2013