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

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(Bench work)
(Bench work)
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==Bench work==
 
==Bench work==
 
# PCR purification
 
# PCR purification
* Followed instructions for Promega Gel and PCR purification kit, using spin column method
+
#* Followed instructions for Promega Gel and PCR purification kit, using spin column method
* Purified experimental BSA PCR sample from [[../23| yesterday]]
+
#* Purified experimental BSA PCR sample from [[../23| yesterday]]
 
# Double-digest
 
# Double-digest
 
{| border="1"
 
{| border="1"
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|0 μL ||5 μL ||0.5 μL ||39.5 μL from purified BSA PCR reaction in step 1 ||2.5μL ||2.5μL
 
|0 μL ||5 μL ||0.5 μL ||39.5 μL from purified BSA PCR reaction in step 1 ||2.5μL ||2.5μL
 
|}
 
|}
*2h @ 37°C
+
#*→2h @ 37°C
 
#<li value="3">Gel purification
 
#<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.
 
#*'''[[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.

Revision as of 14:32, 24 August 2011

BDLlogo notext lr.png AU CHEM-570 Lab Prep <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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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
  2. 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
    • →2h @ 37°C
  1. 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...