Difference between revisions of "Team:UCLA/Notebook/Honeybee Silk/13 July 2015"

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! Component
 
! Component
−
! Volume (out of 25L)
+
! Volume (out of 25uL)
 
|-
 
|-
 
! APEX Taq Red Mastermix
 
! APEX Taq Red Mastermix
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! 10mM primer 10
 
! 10mM primer 10
−
| 2.5uL
+
| 1.25uL
 
|-
 
|-
 
! 10mM primer 11
 
! 10mM primer 11
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| 9uL
 
| 9uL
 
|}
 
|}
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 +
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===Program===
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{| class="wikitable" style="text-align:center; width:600px; height:200px;"
 +
|+
 +
|-
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! Step
 +
! Temperature
 +
! Time
 +
|-
 +
! Initial Denaturation
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| 95C
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| 30s
 +
|-
 +
 +
! Cycles(x30)
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| 95C
 +
| 10s
 +
|-
 +
! Annealing
 +
| 67C
 +
| 20s
 +
|-
 +
! Extension
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| 70C
 +
| 30s
 +
|-
 +
!Final Extension
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| 70C
 +
| 2 min
 +
|-
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! Hold
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|14C
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| Hold
 +
|-
 +
|}
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 +
='''Visualization'''=
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 +
I ran each of the sample on a 1% agarose gel against a 1kB ladder for 20 minutes.
 +
Expected band sizes: ~1000bp
 +
 +
Samples 1 and 3 had bright bands at the appropriate base pair length.
 +
 +
[[File:Gel 2015-07-13.jpg|none|thumb|500px|Gel Image]]
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 +
='''Inoculation'''=
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 +
The remaining 99uL of samples 1 and 3 were inoculated with 5mL LB broth and 5uL 1000X kanamycin, incubated at 37C.
 +
 +
=Testing competency of BL21 cells from 7/12=
 +
*Used puc 19 vector at 50 picograms/ul to test transformation efficiency.
 +
*Added 2 ul of puc 19 to 50 ul of competent BL21 cells
 +
*Used [https://www.neb.com/protocols/1/01/01/high-efficiency-transformation-protocol-c2987 this NEB protocol] to transform with our BL21 cells.
 +
*Plated 10% of the cells on a warm amp plate and put plate in at 4 pm 7/13

Latest revision as of 20:41, 15 July 2015

Colony PCR of 7/12 Transformation

  • The transformation was successful for the 3:1 ligated products plates. I picked 3 colonies off the plate and suspended each in 100uL of ddH2O.
  • Using APEX Taq Red Mastermix


Component Volume (out of 25uL)
APEX Taq Red Mastermix 12.5uL
10mM primer 10 1.25uL
10mM primer 11 1.25uL
Transformed Cells 1uL
Nuclease Free Water 9uL


Program

Step Temperature Time
Initial Denaturation 95C 30s
Cycles(x30) 95C 10s
Annealing 67C 20s
Extension 70C 30s
Final Extension 70C 2 min
Hold 14C Hold

Visualization

I ran each of the sample on a 1% agarose gel against a 1kB ladder for 20 minutes. Expected band sizes: ~1000bp

Samples 1 and 3 had bright bands at the appropriate base pair length.

Gel Image

Inoculation

The remaining 99uL of samples 1 and 3 were inoculated with 5mL LB broth and 5uL 1000X kanamycin, incubated at 37C.

Testing competency of BL21 cells from 7/12

  • Used puc 19 vector at 50 picograms/ul to test transformation efficiency.
  • Added 2 ul of puc 19 to 50 ul of competent BL21 cells
  • Used this NEB protocol to transform with our BL21 cells.
  • Plated 10% of the cells on a warm amp plate and put plate in at 4 pm 7/13