Daniel:Notebook/HiResChrPaint/2013-6-19: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Djacobse
No edit summary
>Djacobse
(Blanked the page)
 
Line 1: Line 1:
=S1 Probe Preparation=


[[Daniel:Notebook/HiResChrPaint|Back to Calendar]]
==PCR amplification==
1. Reaction Table
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="font-size:12pt" align="center"
|style="background-color:#FFFF00" align="center" width="65" height="45" | &nbsp;
| width="65" | 20 nM OligoPool
| width="65" | 2X Kapa SYBR qPCR MM
| width="65" | 100 uM AP1V41U
| width="65" | 100 uM AP2V4
| width="65" | 100 uM AP1V61U
| width="65" | 100 uM AP2V6
| width="66" | 2 mM aa-dUTP
| width="65" | H20
| width="65" | Total Volume
|- style="background-color:#8DB4E2;font-size:12pt" align="center"
| height="15" | per rxn
| align="center" | 0.2
| align="center" | 50
| align="center" | 0.4
| align="center" | 0.4
| align="center" | 0.4
| align="center" | 0.4
| align="center" | 2.5
| align="center" | 45.7
| align="center" | 100
|- style="font-size:12pt" align="center"
| height="30" | V4S1 (12.5x)
| align="center" | 2.5
| align="center" | 625
| align="center" | 5
| align="center" | 5
| align="center" | 0
| align="center" | 0
| align="center" | 31
| align="center" | 581.5
| align="center" | 1250
|- style="font-size:12pt" align="center"
| height="30" | V6S1 (12.5x)
| align="center" | 2.5
| align="center" | 625
| align="center" | 0
| align="center" | 0
| align="center" | 5
| align="center" | 5
| align="center" | 31
| align="center" | 581.5
| align="center" | 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. [[Daniel:Protocols/EtOHPrecip|Ethanol Precipitation]], added 900 uL 100% EtOH, 30 uL 3 M NaOAc, 1 uL glycoblue
6. Incubated 2 hours ([[Daniel:Notebook/HiResChrPaint/2013-6-1|continued 6/1/2013]])

Latest revision as of 17:43, 19 June 2013