Daniel:Notebook/HiResChrPaint/2013-7-22: Difference between revisions

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|- style="font-size:12pt" align="center"
|- style="font-size:12pt" align="center"
| height="30" | V4S3 (12.5x)
| height="30" | V4S2 (12.5x)
| align="center" | 2.5
| align="center" | 2.5
| align="center" | 625
| align="center" | 625
Line 47: Line 47:


|- style="font-size:12pt" align="center"
|- style="font-size:12pt" align="center"
| height="30" | V6S3 (12.5x)
| height="30" | V6S2 (12.5x)
| align="center" | 2.5
| align="center" | 2.5
| align="center" | 625
| align="center" | 625
Line 72: Line 72:
  4. Pooled 3 samples together each (300 uL each, 4 pools per set, 8 total)
  4. Pooled 3 samples together each (300 uL each, 4 pools per set, 8 total)
  5. [[Daniel:Protocols/EtOHPrecip|Ethanol Precipitation]], added 900 uL 100% EtOH, 30 uL 3 M NaOAc, 1 uL glycoblue
  5. [[Daniel:Protocols/EtOHPrecip|Ethanol Precipitation]], added 900 uL 100% EtOH, 30 uL 3 M NaOAc, 1 uL glycoblue
  6. Incubated 4 hours; resuspend in 50 uL nfH20
  6. Incubated 2 hours; resuspend in 50 uL nfH20**
  7. [[Daniel:Protocols/Qiaquick_Column|Column Purification]], elute with 40 uL EB
  7. [[Daniel:Protocols/Qiaquick_Column|Column Purification]], elute with 40 uL EB
  8. Measure concentrations in nanodrop
  8. Measure concentrations in nanodrop
Note(**): It would seem that the time for ethanol precipitation might actually be an important step.  Comparing this trial to previous S2 amplifications, incubating [[Daniel:Notebook/HiResChrPaint/2013-6-1|overnight]], the overnight incubation had much higher yield. So it might do to rethink my pipeline.


===qPCR Results===
===qPCR Results===
[[Image:2013-7-22-S2.png|600px]]


===Nanodrop Results===
===Nanodrop Results===
{| 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" | V4S4A
| width="65" | V4S4B
| width="65" | V4S4C
| width="65" | V4S4D
| width="65" | V6S4A
| width="65" | V6S4B
| width="65" | V6S4C
| width="65" | V6S4D
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | ng/uL
| align="center" | 52.6
| align="center" | 44.5
| align="center" | 52.1
| align="center" | 47.4
| align="center" | 105.7
| align="center" | 111.3
| align="center" | 98.1
| align="center" | 103.3
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | ug in 40 uL
| align="center" | 2.1
| align="center" | 1.8
| align="center" | 2.1
| align="center" | 1.9
| align="center" | 4.2
| align="center" | 4.5
| align="center" | 3.9
| align="center" | 4.1
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | Total ug
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" | 5.8
| align="center" |
| align="center" |
| align="center" |
| align="center" | 12.5
|}
==DpnII Digestion==
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" | V4S4A
| width="65" | V4S4B
| width="65" | V4S4C
| width="65" | V4S4D
| width="65" | V6S4A
| width="65" | V6S4B
| width="65" | V6S4C
| width="65" | V6S4D
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | ng/uL
| align="center" | 52.6
| align="center" | 44.5
| align="center" | 52.1
| align="center" | 47.4
| align="center" | 105.7
| align="center" | 111.3
| align="center" | 98.1
| align="center" | 103.3
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | ug in 35 uL
| align="center" | 2.1
| align="center" | 1.8
| align="center" | 2.1
| align="center" | 1.9
| align="center" | 4.2
| align="center" | 4.5
| align="center" | 3.9
| align="center" | 4.1
|- 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 16 hours; 20 minute heat shock at 65C; [[Daniel:Notebook/HiResChrPaint/2013-7-23|Continued 7/23/2013]]

Latest revision as of 22:30, 23 July 2013

S2 Probe Preparation[edit]

Back to Calendar

Despite mixed reviews from the S4 set, I'm going to go ahead and make the S2 set. Also, I talked with Alan and he and I are going to prep some cells tomorrow. I'll probably go ahead and attempt fish with the S3 set, since that one worked out great. If I have other probes available, I may use them as well, but Alan and I won't be preparing the cells until tomorrow afternoon, which gives me plenty of time to prep a new set.

PCR Amplification[edit]

1. Reaction Table
  20 nM OligoPool 2X Kapa SYBR qPCR MM 100 uM AP1V41U 100 uM AP2V4 100 uM AP1V61U 100 uM AP2V6 2 mM aa-dUTP H20 Total Volume
per rxn 0.2 50 0.4 0.4 0.4 0.4 2.5 45.7 100
V4S2 (12.5x) 2.5 625 5 5 0 0 31 581.5 1250
V6S2 (12.5x) 2.5 625 0 0 5 5 31 581.5 1250
2. Aliquot 100 uL into each of 12 qPCR tubes 
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 x17
    vii. 72C 2 min
    viii. 16C hold
4. Pooled 3 samples together each (300 uL each, 4 pools per set, 8 total)
5. Ethanol Precipitation, added 900 uL 100% EtOH, 30 uL 3 M NaOAc, 1 uL glycoblue
6. Incubated 2 hours; resuspend in 50 uL nfH20**
7. Column Purification, elute with 40 uL EB
8. Measure concentrations in nanodrop

Note(**): It would seem that the time for ethanol precipitation might actually be an important step. Comparing this trial to previous S2 amplifications, incubating overnight, the overnight incubation had much higher yield. So it might do to rethink my pipeline.


qPCR Results[edit]

File:2013-7-22-S2.png

Nanodrop Results[edit]

Sample V4S4A V4S4B V4S4C V4S4D V6S4A V6S4B V6S4C V6S4D
ng/uL 52.6 44.5 52.1 47.4 105.7 111.3 98.1 103.3
ug in 40 uL 2.1 1.8 2.1 1.9 4.2 4.5 3.9 4.1
Total ug       5.8 12.5

DpnII Digestion[edit]

1. Prepare Samples
Sample V4S4A V4S4B V4S4C V4S4D V6S4A V6S4B V6S4C V6S4D
ng/uL 52.6 44.5 52.1 47.4 105.7 111.3 98.1 103.3
ug in 35 uL 2.1 1.8 2.1 1.9 4.2 4.5 3.9 4.1
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 7/23/2013