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,  freeze dried o/n.

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

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 ABM Good Cat.No.: G009

make serial dilution

	Taq concentration	500 ug/ml	50 ug/ml	5 ug/ml
1		T
2			T
3				T
4		S
5			S
6				S
7		U
8			U
9				U
10	Control, 5 units/ul Taq from

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~

 

PCR Mix 1 (with template)

1360 ng/ul template, 2489 pMiniT-TREC-ACTB-ALB	2 ul
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)

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

Fig 1.

Activation Time Test

PCR reaction: 20 ul PCR Mix 1, 0.5 ul of 5 ug/ml Taq 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~

Fig 2

Taq	T	S	U	C	T	S	U	C
Activation Time	5	5	5	5	30'	30'	30'	30'

Amount of Template

	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

1/6/17 number of PCR cycles

	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

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++, 

100 ul 10 mM dNTP

ddH2O to 4 ml

Primers

qPCR QC

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


TREC_HDimer_R
AGGGCCCGTGCCAGCTGCAG

LENGTH	20
GCCONTENT	75 %
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 %
MELT TEMP	60 ºC

Actin_HDimer_R:
5'-GCTCATTGTAGAAGTACTTCAGGG -3'

LENGTH	24
GCCONTENT	45.8 %
MELT TEMP	55 ºC

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

HDimer_F3
AGCTCGTGAAACACAAGCCC

LENGTH	20
GCCONTENT	55 %
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

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

</pre>

ALB2-SYBR-R	CAGCCTTGCAGCACTTCTC
hsjTREC_QC_R	TGCCTATGCATCACCGTG
hsjTREC_QC_F	CACATCCCTTTCAACCATGC
HDimer_F3	AGCTCGTGAAACACAAGCCC
Actin_HDimer_R	GCTCATTGTAGAAGTACTTCAGGG
Actin_HDimer_F	GGGCTTCTTGTCCTTTCCTTCCC
TREC_HDimer_R	AGGGCCCGTGCCAGCTGCAG
TREC_HDimer_F	CACATCCCTTTCAACCATGCTG

F	R	Size
HDimer_F3	Actin_HDimer_R	455

  

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)

 .