Difference between revisions of "Team:UCLA/Notebook/Honeybee Silk/14 July 2015"

(→‎Double Digestion of Purified PCR Product)
(→‎Calculating Competency)
 
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Both samples were sent in for sequencing.
 
Both samples were sent in for sequencing.
  
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='''PCR of Lac promoter/SIlk/Spycatcher'''=
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='''PCR of Lac promoter/Silk/Spycatcher'''=
  
 
*Using primer 3 and 4 that contain overhangs with the Biobrick prefix and suffix.
 
*Using primer 3 and 4 that contain overhangs with the Biobrick prefix and suffix.
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| Hold
 
| Hold
 
|}
 
|}
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*Ran the digestion on a gel (all 50 ul) and compared to NEB 1 kb ladder.  There was a nice clean band at the appropriate size.
 +
[[File:Digest Gel 2015-07-16 at 5.15.51 PM.png|none|thumb|500px|Gel Image]]
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 +
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='''Ligation of SIlk + SpyCatcher into PSB1C3'''=
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*3:1 insert to vector ratio.
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{| class="wikitable" style="text-align:center; width:400px; height:200px;"
 +
|+
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|-
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! Component
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! Volume (out of 50uL)
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|-
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! T4 Ligase Reaction Buffer
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| 2 ul
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|-
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! Backbone (psb1c3)
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| 1uL (50 ng)
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|-
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! Insert (silk + spyC)
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| 10ul (120 ng)
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|-
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! H20
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| 6 ul
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|-
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! Ligase
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| 1uL
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|}
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*Program = 16C overnight and heat inactivate 10 min
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='''Calculating Competency'''=
 
='''Calculating Competency'''=

Latest revision as of 21:47, 24 August 2015

Miniprep

Samples 1 and 3 (inoculated) were miniprepped using the Zymo Plasmid Miniprep Kit.

OD

  1. 128.23 ng/uL
  2. 106.85 ng/uL

Both samples were sent in for sequencing.

PCR of Lac promoter/Silk/Spycatcher

  • Using primer 3 and 4 that contain overhangs with the Biobrick prefix and suffix.


Component Volume (out of 50uL)
5X Q5 Reaction Buffer 10uL
10mM dNTPS 1uL
10mM primer 3 2.5uL
10mM primer 4 2.5uL
Template (Honeybee G-block) 1uL
Q5 High Fidelity DNA Polymerase 0.5uL
Nuclease Free Water 32.5uL

Program

Step Temperature Time
Initial Denaturation 98C 30s
Cycles (x25) 98C 10s
Annealing 72C 20s
Extension 72C 30s
Final Extension 72C 2min
Hold 12C Hold

Gel Visualization and Purification

I ran the PCR product on a 1% agarose gel, expected size ~1600bp

Results: There was a band at the appropriate length and another band, probably the result of non-specific binding. The correct band was extracted and purified using the Qiagen Gel Purification kit.

OD: 88.12ng/uL

Gel Image

Double Digestion of Purified PCR Product

  • The insert will be digested with EcoRI and Pst1 for preparation for ligation into the psb1c3 backbone (already digested with PstI and EcoRI).
Component Volume (out of 50uL)
NEB Buffer 2.1 5uL
EcoRI 1uL
PstI 1uL
Purified Insert 9uL
Nuclease Free Water 34uL

Program

Step Temperature Time
Incubation 37C 1 hour
Heat Inactivation 80C 15 min
Hold 10C Hold
  • Ran the digestion on a gel (all 50 ul) and compared to NEB 1 kb ladder. There was a nice clean band at the appropriate size.
Gel Image


Ligation of SIlk + SpyCatcher into PSB1C3

  • 3:1 insert to vector ratio.
Component Volume (out of 50uL)
T4 Ligase Reaction Buffer 2 ul
Backbone (psb1c3) 1uL (50 ng)
Insert (silk + spyC) 10ul (120 ng)
H20 6 ul
Ligase 1uL
  • Program = 16C overnight and heat inactivate 10 min


Calculating Competency

  • Calculating competency of cells prepared on 7/12.
  • Equation = # colonies / ug DNA plated
  • Using 2 ul of 50pg/ul puc19 vector to transform in to 50 ul
  • Counted 300 colonies
  • plated 1x10^-5 ug (1/10 of transformation reaction).
  • Therefore tranformation efficiency = 3 x 10^7 cfu/ug DNA