Haynes Lab:Notebook/Engineering PC-TFs/2012/11/06: Difference between revisions

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==Summary==
==Summary==
*  
* <u>Gibson Assembly </u>
* Check with PCR


Assembly 1 contains PCR rxns 1,2, and 3. Assembly 2 contains PCR rxns 4,5.
There is no addition of vector this time.
{|border="1" cellpadding="5" cellspacing="0"
|-
! style="background:#efefef;" |
! style="background:#efefef;" | 1
! style="background:#efefef;" | 2
|-
|Gibson Rxn mix
|15 μL
|15 μL
|-
|DNA fragments
|5 μL
|5 μL
|}
{|border="1" cellpadding="5" cellspacing="0"
|-
! scope="col" style="background:#efefef;" | <i>DNA fragments</i>
! scope="col" style="background:#efefef;" | 1
! scope="col" style="background:#efefef;" | 2
|-
|#1
|1.0 μL
|
|-
|#2
|1.0 μL
|
|-
|#3
|1.0 μL
|
|-
|#4
|
|1.0 μL
|-
|#5
|
|1.0 μL
|-
|dH2O
|2.0 μL
|3.0 μL
|-
|<i>Total</i>
|5.0 μL
|5.0 μL
|}
<br><br><br><br>
----
'''PCR Check'''
# Product 1: outer-most primers for whole assembly
# Product 1: Gibson primer pair for hPCD
# Product 2: outer-most primers
# Product 2: Gibson primer pair for hPCD
<br>
{|border="1" cellpadding="5" cellspacing="0"
|-
| <i>Reagent</i> || 1 ||  2 || 3 || 4
|-
| Gibson product || 0.5 μL || 0.5 || 0.5 || 0.5
|-
| primer 1 || 1.0 || 1.0 || 1.0 || 1.0
|-
| primer 2  || 1.0 || 1.0 || 1.0 || 1.0
|-
| 2x GoTaq PCR mix || 14.5 || 14.5 || 14.5 || 14.5
|-
| dH<sub>2</sub>O || 12.0 || 12.0 || 12.0 || 12.0
|}
<br>
<br>
Program:
* 95°C, 3 min.
* [95°C, 30 sec; 57°C, 30 sec; 72°C, 30 sec] 30 cycles
* 72°C, 3 min.
* 4°C ∞


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Revision as of 16:55, 6 November 2012

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Summary

  • Gibson Assembly
  • Check with PCR

Assembly 1 contains PCR rxns 1,2, and 3. Assembly 2 contains PCR rxns 4,5. There is no addition of vector this time.

1 2
Gibson Rxn mix 15 μL 15 μL
DNA fragments 5 μL 5 μL



DNA fragments 1 2
#1 1.0 μL
#2 1.0 μL
#3 1.0 μL
#4 1.0 μL
#5 1.0 μL
dH2O 2.0 μL 3.0 μL
Total 5.0 μL 5.0 μL





PCR Check

  1. Product 1: outer-most primers for whole assembly
  2. Product 1: Gibson primer pair for hPCD
  3. Product 2: outer-most primers
  4. Product 2: Gibson primer pair for hPCD


Reagent 1 2 3 4
Gibson product 0.5 μL 0.5 0.5 0.5
primer 1 1.0 1.0 1.0 1.0
primer 2 1.0 1.0 1.0 1.0
2x GoTaq PCR mix 14.5 14.5 14.5 14.5
dH2O 12.0 12.0 12.0 12.0



Program:

  • 95°C, 3 min.
  • [95°C, 30 sec; 57°C, 30 sec; 72°C, 30 sec] 30 cycles
  • 72°C, 3 min.
  • 4°C ∞


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