User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/08/24: Difference between revisions
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## 50μL pTXB1 double-digest from step 2 + 10μL 6X loading buffer | ## 50μL pTXB1 double-digest from step 2 + 10μL 6X loading buffer | ||
## 50μL BSA PCR 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== | ==Results== | ||
* | * GEL | ||
[[Image: DNAgel_purification_8.24.2011_labeled.jpg | 300px]] | |||
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> | <!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> | ||
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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 <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
ObjectiveRepeat BSA cloning into intein vectors with new, correct 3' primer. Bench work
Results
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