Daniel:Notebook/HiResChrPaint/2013-8-14: Difference between revisions

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=Amino Group Primers=


[[Daniel:Notebook/HiResChrPaint|Back to Calendar]]
So the new primers are in.  We ordered them a few weeks ago.  All the primers have a 5' amino group, which should be compatible with the Alexa-dye coupling system.  Thanks to [[Matt:LabNotes/2013-9-25|Matt's results]] yesterday, I know that when the dye coupling does not work, the Centri-Sep columns yield very little dye.  So, before going nuts on making new probes, I'm going to test the water with a simple comparison.  For sample 1, I'm going to test the dye using the new primer, plus no aa-dUTP.  I'm also going to do a positive control using a standard amplification using the old primers and aa-dUTP.  Finally, I'll run a negative control using old primer and no aa-dUTP.  I'll do this for both V4S3 and V6S3, since I should test both primer sets.
==Amino Primers Test==
===qPCR===
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="81" 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="65" | 50 mM aa-dUTP
| width="65" | H20
| width="65" | Total Volume
|- style="background-color:#8DB4E2;font-size:12pt" align="center"
| height="15" | std 100 uL 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" | 0.1
| align="center" | 46
| align="center" | 100
|- style="font-size:12pt" align="center"
| height="15" | V4S3* (5x)
| align="center" | 1
| align="center" | 250
| align="center" | 2
| align="center" | 2
| align="center" | 0
| align="center" | 0
| align="center" | 0
| align="center" | 245
| align="center" | 500
|- style="font-size:12pt" align="center"
| height="15" | V6S3* (5x)
| align="center" | 1
| align="center" | 250
| align="center" | 0
| align="center" | 0
| align="center" | 2
| align="center" | 2
| align="center" | 0
| align="center" | 245
| align="center" | 500
|- style="font-size:12pt" align="center" valign="bottom"
| height="15" | V6S3 (5x)
| align="center" | 1
| align="center" | 250
| align="center" | 0
| align="center" | 0
| align="center" | 2
| align="center" | 2
| align="center" | 0.5
| align="center" | 244.5
| align="center" | 500
|- style="font-size:12pt" align="center" valign="bottom"
| height="15" | V6S3- (5x)
| align="center" | 1
| align="center" | 250
| align="center" | 0
| align="center" | 0
| align="center" | 2
| align="center" | 2
| align="center" | 0
| align="center" | 245
| align="center" | 500
|}
2. Aliquot 100 uL into each of 5 qPCR tubes (20 in total)
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 and 2 samples together (300, 200 uL each)

Latest revision as of 20:15, 26 September 2013