Editing Taq for Real Time PCR (section)

== 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/>
|}