Editing Taq for Real Time PCR (section)

== Primer Dimer Aanalysis ==

'''PCR Mix without Taq & Primers'''

Template: 1 ul of 1 mg/ml maxiprep DNA.

400 ul of 10X PCR Buffer with Mg++,&nbsp;

100 ul 10 mM dNTP

ddH2O to 4 ml

'''Primers'''
<pre>qPCR QC

TREC_HDimer_F
5'-CACATCCCTTTCAACCATGCTG -3'
LENGTH	22
GCCONTENT	50&nbsp;%
MELTTEMP	56.6 ºC


TREC_HDimer_R
AGGGCCCGTGCCAGCTGCAG

LENGTH	20
GCCONTENT	75&nbsp;%
MELT TEMP	68.2 ºC

Delta G:  -3.55 kcal/mole   Base Pairs3
5' CACATCCCTTTCAACCATGCTG
                      |||                 
3'                    GACGTCGACCGTGCCCGGGA


actin
Actin_HDimer_F:
GGGCTTCTTGTCCTTTCCTTCCC

LENGTH	23
GCCONTENT	56.5&nbsp;%
MELT TEMP	60 ºC

Actin_HDimer_R:
5'-GCTCATTGTAGAAGTACTTCAGGG -3'

LENGTH	24
GCCONTENT	45.8&nbsp;%
MELT TEMP	55 ºC

DeltaG:  -6.14 kcal/mole   Base Pairs3
5'GGGCTTCTTGTCCTTTCCTTCCC
                      |||                    
3'                    GGGACTTCATGAAGATGTTACTCG 5'

HDimer_F3
AGCTCGTGAAACACAAGCCC

LENGTH	20
GCCONTENT	55&nbsp;%
MELT TEMP	58.2 ºC
Delta G:  -6.14 kcal/mole   Base Pairs3

5' AGCTCGTGAAACACAAGCCC
                    |||                 
3'                  GGGACTTCATGAAGATGTTACTC 5'

tatagcatatgaatgctgcacagaatccttggaattcctgtcaacaaaggtgatgc
cacatccctttcaaccatgctgacacctctggtttttgtaaaggtgcccactcctgtgcacggtgatgcataggcacctgcaccccgtgcctaaaccctgcagctggcacgggccctgtctgctcttcattcaccgttctcacgagttgcaataagttcagccctccatgtcacactgtgttttccatcGGATCCTTGCTGTCATCTCTTGTGGGCTGTAATCATCGTCTAGGCTTAAGAGTAATATTGCAAAACCTGTCATGCCCACACAAATCTCTCCCTGGCATTGTTGTCTTTGCAGATGTCAGTGAAAGAGAACCAGCAGCTCcacttgttg

455 bp
HDimer_F3
agctcgtgaaacacaagccc
aaggcaacaaaagagcaactgaaagctgttatggatgatttcgcagcttttgtagagaagtgctgcaaggctgccATGGATGATGATATCGCCGCGCTCGTCGTCGACAACGGCTCCGGCATGTGCAAGGCCGGCTTCGCGGGCGACGATGCCCCCCGGGCCGTCTTCCCCTCCATCGTGGGGCGCCCCAGGCACCAGGtaggggagctggctgggtggggcagccccgggagcgggcgggaggcaagggcgctttctctgcacaggagcctcccggtttccggggtgggggctgcgcccgtgGGTACCctcagggcttcttgtcctttccttcccagGGCGTGATGGTGGGCATGGGTCAGAAGGATTCCTATGTGGGCGACGAGGCCCAGAGCAAGAGAGGCATCCTCA
Actin_HDimer_R
CCCTGAAGTACttctacaatgagc


</pre>

'''TREC Assay Probes'''

hALB-probe3 5' JOE NHS/ZEN/3' IBFQ Length 24 Purification HPLC Sequence 56-JOEN/CCCACACAA/ZEN/ATCTCTCCCTGGCAT/3IABkFQ/

hBetaActin-probe3 5' JOE NHS/ZEN/3' IBFQ Length 23 Purification HPLC Sequence 56-JOEN/TGATGGTGG/ZEN/GCATGGGTCAGAAG/3IABkFQ/

hsjTREC-probe 5' 6-FAM/ZEN/3' IBFQ Length 30 Purification HPLC Sequence 56-FAM/ACACCTCTG/ZEN/GTTTTTGTAAAGGTGCCCACT/3IABkFQ/

Forward CACATCCCTTTCAACCATGC Probe TGTAAAGGTGCCCACTCCTGTGC Reverse TGCCTATGCATCACCGTG

cagccttgcagcacttctc

&lt;/pre&gt;

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| <br/>
| <br/>
|-
| ALB2-SYBR-R<br/>
| CAGCCTTGCAGCACTTCTC<br/>
|-
| hsjTREC_QC_R<br/>
| TGCCTATGCATCACCGTG<br/>
|-
| hsjTREC_QC_F<br/>
| CACATCCCTTTCAACCATGC<br/>
|-
| HDimer_F3<br/>
| AGCTCGTGAAACACAAGCCC<br/>
|-
| Actin_HDimer_R<br/>
| GCTCATTGTAGAAGTACTTCAGGG<br/>
|-
| Actin_HDimer_F<br/>
| GGGCTTCTTGTCCTTTCCTTCCC<br/>
|-
| TREC_HDimer_R<br/>
| AGGGCCCGTGCCAGCTGCAG<br/>
|-
| TREC_HDimer_F<br/>
| CACATCCCTTTCAACCATGCTG<br/>
|}







{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
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| F
| R
| Size
|-
| <pre>HDimer_F3</pre>

| <pre>Actin_HDimer_R</pre>

| 455
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&nbsp;&nbsp;

'''PCR'''

for each reaction:

13 ul PCR buffer mix

0.5 ul 10 uM F primer

0.5 ul 10 uM R primer

0.5 ul Taq