Daniel:Notebook/HiResChrPaint/2013-6-20: Difference between revisions

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| align="center" |  
| align="center" |  
| align="center" |  
| align="center" | 4.896
| align="center" | 4.896*
| align="center" |  
| align="center" |  
| align="center" |  
| align="center" |  
| align="center" |  
| align="center" |  
| align="center" | 14.38
| align="center" | 14.38**


|}
|}
Notes: *1.7x yield (in ug) of qPCR of V4S1 using 800 uL starting material; ** 2.1x yield (in ug) of qPCR of V6S1 using 800 uL starting material
===Discussion===
Results were not so good, although they should have been expected (see the notes).  From the results I am skeptical of my final yields once all is
said and done.  Therefore, I will proceed to DpnII digestion with caution.
==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="62" 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" | 30.7
| align="center" | 31.1
| align="center" | 31.5
| align="center" | 29.1
| align="center" | 90
| align="center" | 87.7
| align="center" | 91.9
| align="center" | 89.9
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | ug in 35 uL
| align="center" | 1.1
| align="center" | 1.1
| align="center" | 1.1
| align="center" | 1.0
| align="center" | 3.2
| align="center" | 3.1
| align="center" | 3.2
| align="center" | 3.1
|- 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/2013-6-21|Continued]] 6/21/2013]]

Latest revision as of 00:21, 21 June 2013

S1 Probe Preparation[edit]

Back to Calendar

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
    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

qPCR Results[edit]

File:V4S1-062013.png

Nanodrop Results[edit]

Sample V4S1A V4S1B V4S1C V4S1D V6S1A V6S1B V6S1C V6S1D
ng/uL 30.7 31.1 31.5 29.1 90 87.7 91.9 89.9
ug in 40 uL 1.228 1.244 1.26 1.164 3.6 3.508 3.676 3.596
Total ug       4.896*       14.38**

Notes: *1.7x yield (in ug) of qPCR of V4S1 using 800 uL starting material; ** 2.1x yield (in ug) of qPCR of V6S1 using 800 uL starting material

Discussion[edit]

Results were not so good, although they should have been expected (see the notes).  From the results I am skeptical of my final yields once all is
said and done.  Therefore, I will proceed to DpnII digestion with caution.

DpnII Digestion[edit]

1. Prepare Samples
Sample V4S1A V4S1B V4S1C V4S1D V6S1A V6S1B V6S1C V6S1D
ng/uL 30.7 31.1 31.5 29.1 90 87.7 91.9 89.9
ug in 35 uL 1.1 1.1 1.1 1.0 3.2 3.1 3.2 3.1
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 6/21/2013]]