Daniel:Notebook/HiResChrPaint/2013-8-2: Difference between revisions

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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" | V4S1A
| width="65" | V4S1B
| width="65" | V4S1C
| width="65" | V4S1D
| width="65" | V6S1A
| width="65" | V6S1B
| width="65" | V6S1C
| width="65" | V6S1D
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | ng/uL
| align="center" | 96.8
| align="center" | 92.3
| align="center" | 90.1
| align="center" | 97.2
| align="center" | 141.6
| align="center" | 148.1
| align="center" | 142
| align="center" | 136.3
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | ug in 35 uL
| align="center" | 3.4
| align="center" | 3.2
| align="center" | 3.2
| align="center" | 3.4
| align="center" | 5.0
| align="center" | 5.2
| align="center" | 5.0
| align="center" | 4.8
|- 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 2 hours; 20 minute heat shock at 65C
3. [[Daniel:Protocols/Qiaquick_Column|Column purification]], elute with 30 uL EB
4. Measure in Nanodrop
===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" | V4S1A
| width="65" | V4S1B
| width="65" | V4S1C
| width="65" | V4S1D
| width="65" | V6S1A
| width="65" | V6S1B
| width="65" | V6S1C
| width="65" | V6S1D
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | ng/uL
| align="center" | 88.2
| align="center" | 87.1
| align="center" | 77.4
| align="center" | 94.7
| align="center" | 129.9
| align="center" | 142.6
| align="center" | 139.6
| align="center" | 132.3
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | ug in 30 uL
| align="center" | 2.6
| align="center" | 2.6
| align="center" | 2.3
| align="center" | 2.8
| align="center" | 3.9
| align="center" | 4.3
| align="center" | 4.2
| align="center" | 4.0
|- 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" | 10.4
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" | 16.3
|}
===TBE Gel===
{{GelLanes|Lane3=10 bp ladder|Lane4=V4S1A|Lane5=V4S1B|Lane6=V4S1C||Lane7=V4S1D|Lane8=V6S1A|Lane9=V6S1B|Lane10=V6S1C|Lane11=V6S1D}}
'''Gel Image'''

Latest revision as of 00:37, 3 August 2013

S1 Probe Preparation[edit]

Back to Calendar

Armed with a new bottle of water (seriously...), I'm now ready to amplify the S1 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
V4S1 (12.5x) 2.5 625 5 5 0 0 31 581.5 1250
V6S1 (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. Pool 3 samples together each (300 uL each, 4 pools per set, 8 total)
5. Column Purification, elute with 40 uL EB
6. Measure concentrations in nanodrop

qPCR Results[edit]

Nanodrop Results[edit]

Sample V4S1A V4S1B V4S1C V4S1D V6S1A V6S1B V6S1C V6S1D
ng/uL 96.8 92.3 90.1 97.2 141.6 148.1 142 136.3
ug in 40 uL 3.9 3.7 3.6 3.9 5.7 5.9 5.7 5.5
Total ug       15.1       22.7

DpnII Digestion[edit]

1. Prepare Samples
Sample V4S1A V4S1B V4S1C V4S1D V6S1A V6S1B V6S1C V6S1D
ng/uL 96.8 92.3 90.1 97.2 141.6 148.1 142 136.3
ug in 35 uL 3.4 3.2 3.2 3.4 5.0 5.2 5.0 4.8
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 2 hours; 20 minute heat shock at 65C
3. Column purification, elute with 30 uL EB
4. Measure in Nanodrop

Nanodrop Results[edit]

Sample V4S1A V4S1B V4S1C V4S1D V6S1A V6S1B V6S1C V6S1D
ng/uL 88.2 87.1 77.4 94.7 129.9 142.6 139.6 132.3
ug in 30 uL 2.6 2.6 2.3 2.8 3.9 4.3 4.2 4.0
Total ug       10.4       16.3

TBE Gel[edit]

Lane 1 2 3 4 5 6 7 8 9 10 11 12
Sample 10 bp ladder V4S1A V4S1B V4S1C V4S1D V6S1A V6S1B V6S1C V6S1D

Gel Image