Difference between revisions of "Team:UCLA/Notebook/Honeybee Silk/10 July 2015"

(→‎Cloning Honeybee Silk into Pet 24a)
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=Cloning Honeybee Silk into Pet 24a=
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='''Double Digestion and Cleanup of pET24a'''=
 
*Sequencing results checked out for miniprepped pet 24a plasmid.
 
*Sequencing results checked out for miniprepped pet 24a plasmid.
−
*Double digested PET 24a with BamH1 and Nde1 restriction enzymes
+
*I will be using the first sample from 7/9  (OD: 231.26ng/uL)
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*Ran on gel, excised, and purified
+
 
 +
==Digestion Reaction==
 +
 
 +
{| class="wikitable" style="text-align:center; width:400px; height:200px;"
 +
|+
 +
|-
 +
! Component
 +
! Volume (out of 50uL)
 +
|-
 +
! NEB 2.1 Buffer
 +
| 5uL
 +
|-
 +
! BamHI
 +
| 1uL
 +
|-
 +
! NdeI
 +
| 1uL
 +
|-
 +
! pET24a (Miniprepped)
 +
| 5uL
 +
|-
 +
 
 +
!Nuclease Free Water
 +
| 38uL
 +
|}
 +
 
 +
===Program===
 +
 
 +
{| class="wikitable" style="text-align:center; width:400px; height:200px;"
 +
|+
 +
|-
 +
! Step
 +
! Temperature
 +
!Time
 +
|-
 +
! Incubation
 +
| 37C
 +
| 1 hour
 +
|-
 +
! Heat Inactivation
 +
| 80C
 +
| 15 min
 +
|-
 +
!Hold
 +
| 10C
 +
| Hold
 +
|}
 +
 
 +
I ran the sample on a 1% agarose gel against a 1kB, the appropriate band length was present (5310 bp).
 +
 
 +
(picture, do this asap)
 +
 
 +
The band was excised from the gel and purified along with the collected gel sample of the honeybee insert from 7/8 using the Qiagen Gel Purification kit.
 +
 
 +
OD:
 +
Honeybee silk: 28.4ng/uL
 +
pET24a: 23.6
 +
 
 +
 
 +
='''Ligation'''=
 
*Ran ligation reaction with double digested pet 24a and honeybee sequenced
 
*Ran ligation reaction with double digested pet 24a and honeybee sequenced
 +
 
{| class="wikitable" style="text-align:center; width:400px; height:200px;"
 
{| class="wikitable" style="text-align:center; width:400px; height:200px;"
 
|+  
 
|+  

Revision as of 04:10, 11 July 2015

Double Digestion and Cleanup of pET24a

  • Sequencing results checked out for miniprepped pet 24a plasmid.
  • I will be using the first sample from 7/9 (OD: 231.26ng/uL)

Digestion Reaction

Component Volume (out of 50uL)
NEB 2.1 Buffer 5uL
BamHI 1uL
NdeI 1uL
pET24a (Miniprepped) 5uL
Nuclease Free Water 38uL

Program

Step Temperature Time
Incubation 37C 1 hour
Heat Inactivation 80C 15 min
Hold 10C Hold

I ran the sample on a 1% agarose gel against a 1kB, the appropriate band length was present (5310 bp).

(picture, do this asap)

The band was excised from the gel and purified along with the collected gel sample of the honeybee insert from 7/8 using the Qiagen Gel Purification kit.

OD: Honeybee silk: 28.4ng/uL pET24a: 23.6


Ligation

  • Ran ligation reaction with double digested pet 24a and honeybee sequenced
Component 3:1 insert:vector) Volume (out of 20uL)
T4 Ligase Buffer 2 ul
Pet 24a backbone (23.6 ng/ul) 2.54 ul
SIlk insert 28.4 ng/ul 1.12
T4 Ligase 1 ul
Water 13.34
Component 5:1 insert to vector Volume (out of 20uL)
T4 Ligase Buffer 2 ul
Pet 24a backbone (23.6 ng/ul) 2.54 ul
SIlk insert 28.4 ng/ul 1.9
T4 Ligase 1 ul
Water 12.56

Running ligation reaction overnight 16 C starting at 6:45 pm 7/10