Editing Taq for Real Time PCR

==  ==

== Taq Purification ==

Taq from 1032.



== Chemical Treatment ==

1 mg Taq protein ~3 mg/ml, add 5% sucrose (S), or trehalose (T), dissolve, &nbsp;freeze dried o/n.

Resuspend Taq in 950 ul tert-butyl methyl ether (tBME), add 50 ul &nbsp;citraconic anhydride (final conc 5%)., react at 37 oC for 5 hours shaking,&nbsp;

Wash 3 times with 1 ml tBME, air dry o/n inside a chemical hood.

Add 300 ul H2O, dto dissolve on ice.

Taq without sucrose or trehalose is diluted 1X of storage buffer. 1.27 mg/ml

Diluted chemical treated Taq to 1.27 mg/ml, store at -20oC



== PCR QC ==

1 Marke serial dilution. Assuming 1 mg Taq is 90,000 units.

T: Trehalose

S: Sucrose

U: Untrrated

C: Reference Taq, 5 units/ul Taq from&nbsp;ABM Good Cat.No.: G009

make serial dilution

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| <br/>
| Taq concentration
| 500 ug/ml<br/>
| 50 ug/ml
| 5 ug/ml
|-
| 1
| <br/>
| T
| <br/>
| <br/>
|-
| 2
| <br/>
| <br/>
| T
| <br/>
|-
| 3
| <br/>
| <br/>
| <br/>
| T
|-
| 4
| <br/>
| S
| <br/>
| <br/>
|-
| 5
| <br/>
| <br/>
| S
| <br/>
|-
| 6
| <br/>
| <br/>
| <br/>
| S
|-
| 7
| <br/>
| U
| <br/>
| <br/>
|-
| 8
| <br/>
| <br/>
| U
| <br/>
|-
| 9
| <br/>
| <br/>
| <br/>
| U
|-
| 10
| Control, 5 units/ul Taq from
| <br/>
| <br/>
| <br/>
|}



PCR reaction: 20 ul PCR Mix 1, 1 ul diluted Taq 1~9, or Control (10)

PCR: 95 oC 5 min, 30 cycles of 95 oC 30', 55 oC 30', 72 oC 1 min, then 72 oC 3 min, 4 oC~



&nbsp;

'''PCR Mix 1 (with template)'''

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| 1360 ng/ul template,&nbsp;2489 pMiniT-TREC-ACTB-ALB<br/>
| 2 ul&nbsp;
|-
| Primer F: 8_3_16 hsjTREC-F
| 50 ul
|-
| Primer R: human_beta_actin_SYBR-R4
| 50 ul
|-
| 10 X PCR buffer with Mg2+
| 125 ul
|-
| 10 mM dNTP
| 50 ul
|-
| H2O
| 975 ul
|}

'''PCR Mix 2 (without template)'''

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| Primer F: 8_3_16 hsjTREC-F
| 50 ul
|-
| Primer R: human_beta_actin_SYBR-R4
| 50 ul
|-
| 10 X PCR buffer with Mg2+
| 125 ul
|-
| 10 mM dNTP
| 50 ul
|-
| H2O
| 975 ul
|}





[[File:Taq chemical modification 1 4 16.jpg|RTENOTITLE]]

Fig 1.





'''Activation Time Test'''

PCR reaction: 20 ul PCR Mix 1, 0.5&nbsp;ul of 5 ug/ml Taq&nbsp;or Control (10)

PCR: 95 oC 5 min or 30 sec, 30 cycles of 95 oC 30', 55 oC 30', 72 oC 1 min, then 72 oC 3 min, 4 oC~





[[File:Taq chemical modification activation time 1 4 16.jpg|RTENOTITLE]]

Fig 2

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
|-
| Taq
| T
| S
| U
| C
| T
| S
| U
| C
|-
| Activation Time
| 5
| 5
| 5
| 5
| 30'
| 30'<br/>
| 30'<br/>
| 30'<br/>
|}





==  ==

'''Amount of Template'''

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| <br/>
| PCR Mix 1
| PCR Mix 2
| Taq, 5 ug/ml
|-
| 1
| 1
| 19
| 0.5 ul of T
|-
| 2
| 1
| 19
| S
|-
| 3
| 1
| 19
| U
|-
| 4
| 1
| 19
| C
|-
| 5
| 5
| 15
| T
|-
| 6
| 5
| 15
| S
|-
| 7
| 5
| 15
| U
|-
| 8
| 5
| 15
| C
|-
| 9
| 10
| 10
| T
|-
| 10
| 10
| 10
| S
|-
| 11
| 10
| 10
| U
|-
| 12
| 10
| 10
| C
|-
| 13
| 15
| 5
| T
|-
| 14
| 15
| 5
| S
|-
| 15
| 15
| 5
| U
|-
| 16
| 15
| 5
| C
|-
| 17
| 20
| 0
| T
|-
| 18
| 20
| 0
| S
|-
| 19
| 20
| 0
| U
|-
| 20
| 20
| 0
| C
|}

==  ==

== [[File:Taq QC Template conc 1 5 17.jpg|RTENOTITLE]] ==

1/6/17 number of PCR cycles

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| <br/>
| Cycles
| Enzyme
|-
| 1
| 20
| T
|-
| 2
| 20
| S
|-
| 3
| 20
| U
|-
| 4
| 20
| C
|-
| 5
| 25
| T
|-
| 6
| 25
| S
|-
| 7
| 25
| U
|-
| 8
| 25
| B
|-
| 9
| 30
| T
|-
| 10
| 30
| S
|-
| 11
| 30
| U
|-
| 12
| 30
| B
|-
| 13
| 35
| T
|-
| 14
| 35
| S
|-
| 15
| 35
| U
|-
| 16
| 35
| B
|-
| 17
| 40
| T
|-
| 18
| 40
| S
|-
| 19
| 40
| U
|-
| 20
| 40
| B
|}

[[File:Taq chemical modification QC 1 6 16.jpg|RTENOTITLE]]

==  ==

== 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;"
|-
| F
| R
| Size
|-
| <pre>HDimer_F3</pre>

| <pre>Actin_HDimer_R</pre>

| 455
|-
| <br/>
| <br/>
| <br/>
|-
| <br/>
| <br/>
| <br/>
|-
| <br/>
| <br/>
| <br/>
|-
| <br/>
| <br/>
| <br/>
|-
| <br/>
| <br/>
| <br/>
|-
| <br/>
| <br/>
| <br/>
|}

&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

== PCR Enhancer (additives) ==

ethylene glycol

1,2-propanediol.

TMAC (Tetramethyl ammonium chloride)

&nbsp;.


[[Category:QPCR RT-PCR]]