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

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{{Undergrad Notebook 1}}
==Objective==
Repeat BSA cloning into intein vectors with new, correct 3' primer.
 
==Bench work==
# 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
|}
:*&rarr;2h @ 37°C
#<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
# Spun down 4 x 500mL growths from [[../23 | yesterday]] (10 minutes at 3810rpm and 4°C)
#* Resuspended pMAL-pIII in 100mL 30mM TrisCl 20% sucrose w/v pH 8.0 then placed in freezer @ -20°C°C
#* Resuspended pMXB10 in 25 mL 20mM TrisCl 500mM NaCl pH 8.5 then quick froze with liquid nitrogen → freezer @ -80°C
# After gel purification, added 6μL 10x phosphatase buffer, 3μL dH<sub>2</sub>O, and 1μL phosphatase to the 50mL the two plasmids were eluted into.
#* Heat inactivated phosphatase with 5 minutes at 65°C
{| border="1"
|-
!Tube !!sterile H<sub>2</sub>O !!10X ligase buffer !! BSA !!plasmid !!T4 DNA ligase
|-
!pTXB1 + BSA
|13μL || 5μL || 20μL || 10μL || 2μL
|-
!pTXB1 (-)
|33μL || 5μL || 0μL || 10μL || 2μL 
|-
!pTXB1.His + BSA
|13μL || 5μL || 20μL || 10μL || 2μL
|-
!pTXB1.His (-)
|33μL || 5μL || 0μL || 10μL || 2μL
|}
 
==Results==
* GEL
[[Image: DNAgel_purification_8.24.2011_labeled.jpg | 300px]]
 


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Revision as of 11:39, 28 August 2011

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
  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
  3. Spun down 4 x 500mL growths from yesterday (10 minutes at 3810rpm and 4°C)
    • Resuspended pMAL-pIII in 100mL 30mM TrisCl 20% sucrose w/v pH 8.0 then placed in freezer @ -20°C°C
    • Resuspended pMXB10 in 25 mL 20mM TrisCl 500mM NaCl pH 8.5 then quick froze with liquid nitrogen → freezer @ -80°C
  4. After gel purification, added 6μL 10x phosphatase buffer, 3μL dH2O, and 1μL phosphatase to the 50mL the two plasmids were eluted into.
    • Heat inactivated phosphatase with 5 minutes at 65°C
Tube sterile H2O 10X ligase buffer BSA plasmid T4 DNA ligase
pTXB1 + BSA 13μL 5μL 20μL 10μL 2μL
pTXB1 (-) 33μL 5μL 0μL 10μL 2μL
pTXB1.His + BSA 13μL 5μL 20μL 10μL 2μL
pTXB1.His (-) 33μL 5μL 0μL 10μL 2μL

Results

  • GEL


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