Haynes Lab:Notebook/Engineering PC-TFs/2014/11/10: Difference between revisions
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| colspan="2"| | | colspan="2"| | ||
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* DNA Part to Oligo Bridge is 1:10 (DNA part ratio is 1:1) | * DNA Part to Oligo Bridge is 1:10 (DNA part ratio is 1:1) | ||
* X ng insert = (bp inset / bp vector) x 1 x 50 ng vector | * X ng insert = (bp inset / bp vector) x 1 x 50 ng vector | ||
* X ng BL01& | * X ng BL01&5 = (2520bp/5197bp)*1*50ng vector = 24.24ng BL01&9 | ||
* Volume BL01& | * Volume BL01&5 = 24.24ng*(1μL/10ng) = 2.424μL | ||
* Volume CMV/MV9 = 50ng*(1μL/33ng) = 1.28μL | * Volume CMV/MV9 = 50ng*(1μL/33ng) = 1.28μL | ||
* 2:1 vector to insert use 2.56μL vector. | * 2:1 vector to insert use 2.56μL vector. | ||
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| || <u>Ligation</u> || <u>Negative Control</u> | | || <u>Ligation</u> || <u>Negative Control</u> | ||
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| Insert DNA (BL01, BL05 respectively) || | | Insert DNA (BL01, BL05 respectively) || 2.5 μL || '''none''' | ||
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| Vector DNA (50 ng) || | | Vector DNA (50 ng) || 1.3 μL || same | ||
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| 10x Biolabs T4 Ligase Buffer || 2.0 μl || same | | 10x Biolabs T4 Ligase Buffer || 2.0 μl || same | ||
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| Biolabs T4 Ligase || 1.0 μl || same | | Biolabs T4 Ligase || 1.0 μl || same | ||
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| dH<sub>2</sub>O || | | dH<sub>2</sub>O || 13.2 μL || 15.7 μL | ||
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| || 20.0 μL total || same | | || 20.0 μL total || same | ||
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*DH5α-T cells were thawed in ice. | *DH5α-T cells were thawed in ice. | ||
* | *Clean 1.5mL tubes were setup and labeled. | ||
*60μL of thawed cells were mixed and then transferred into each | *60μL of thawed cells were mixed and then transferred into each tube. | ||
*The ligation products were then transferred into their respective tubes and flicked to mix. Set back on ice. | *The ligation products were then transferred into their respective tubes and flicked to mix. Set back on ice. | ||
* | *BL05 in V0120 was used as a positive control; 1μL of this plasmid was added to 19μL dH<sub>2</sub>O to equal the other two products' 20μL total volume. Added to the fifth tube with 60μL thawed cells. | ||
*All tubes incubated on ice for 35 minutes. | *All tubes incubated on ice for 35 minutes. | ||
*All tubes were heat shocked on the heat block at 42°C for 30 seconds and then placed back on ice for 2 minutes. | *All tubes were heat shocked on the heat block at 42°C for 30 seconds and then placed back on ice for 2 minutes. |
Latest revision as of 00:32, 27 September 2017
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Summary
LCR Calculations
Long transformation protocol used for LCR reaction mixture.
Long Transformation
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