User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/08/24: Difference between revisions

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==Objective==
==Objective==
Add today's objective(s) here...
Repeat BSA cloning into intein vectors with new, correct 3' primer.


==Bench work==
==Bench work==
# Add experimental record here...
# PCR purification
* Followed instructions for Promega Gel and PCR purification kit, using spin column method
* Purified experimental BSA PCR sample from [[../23| yesterday]]
# Double-digest
{| border="1"
|-
!Tube !!sterile H<sub>2</sub>O !!10X NEB2 !!100X BSA !!DNA !!NheI (10 kU/mL) !!SapI (2 kU/mL
|-
!pTXB1His
|14.5 μL ||5 μL ||0.5 μL ||25 μL of 102 μg/mL ||2.5μL ||2.5μL
|-
!pTXB1
|14.5 μL ||5 μL ||0.5 μL ||25 μL of 120 μg/mL ||2.5μL ||2.5μL
|-
!BSA PCR
|0 μL ||5 μL ||0.5 μL ||39.5 μL from purified BSA PCR reaction in step 1 ||2.5μL ||2.5μL
|}
#<li value="3">Gel purification
#*'''[[User:Kathryn Muratore|Kathryn Muratore]] 17:28, 24 August 2011 (EDT)''':I forgot to phosphatase the vectors before loading the gel, so we will phosphatase after purification.
## 50μL pTXB1His double-digest from step 2 + 10μL 6X loading buffer
## 10μL 1KB ladder
## skip
## 50μL pTXB1 double-digest from step 2 + 10μL 6X loading buffer
## 50μL BSA PCR double-digest from step 2 + 10μL 6X loading buffer
#


==Results==
==Results==

Revision as of 14:28, 24 August 2011

AU CHEM-570 Lab Prep <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

Objective

Repeat BSA cloning into intein vectors with new, correct 3' primer.

Bench work

  1. PCR purification
  • Followed instructions for Promega Gel and PCR purification kit, using spin column method
  • Purified experimental BSA PCR sample from yesterday
  1. Double-digest
Tube sterile H2O 10X NEB2 100X BSA DNA NheI (10 kU/mL) SapI (2 kU/mL
pTXB1His 14.5 μL 5 μL 0.5 μL 25 μL of 102 μg/mL 2.5μL 2.5μL
pTXB1 14.5 μL 5 μL 0.5 μL 25 μL of 120 μg/mL 2.5μL 2.5μL
BSA PCR 0 μL 5 μL 0.5 μL 39.5 μL from purified BSA PCR reaction in step 1 2.5μL 2.5μL
  2. Gel purification
    • Kathryn Muratore 17:28, 24 August 2011 (EDT):I forgot to phosphatase the vectors before loading the gel, so we will phosphatase after purification.
    1. 50μL pTXB1His double-digest from step 2 + 10μL 6X loading buffer
    2. 10μL 1KB ladder
    3. skip
    4. 50μL pTXB1 double-digest from step 2 + 10μL 6X loading buffer
    5. 50μL BSA PCR double-digest from step 2 + 10μL 6X loading buffer

Results

  • Add results here...


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