Haynes Lab:Notebook/Engineering PC-TFs/2014/11/10: Difference between revisions
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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> | ||
|- | |- | ||
| Insert DNA (BL01, BL05 respectively) || | | Insert DNA (BL01, BL05 respectively) || 2.5 μL || '''none''' | ||
|- | |- | ||
| Vector DNA (50 ng) || | | Vector DNA (50 ng) || 1.3 μL || same | ||
|- | |- | ||
| 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 | ||
|- | |- | ||
| dH<sub>2</sub>O || | | dH<sub>2</sub>O || 13.2 μL || 15.7 μL | ||
|- | |- | ||
| || 20.0 μL total || same | | || 20.0 μL total || same |
Revision as of 22:14, 22 November 2014
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Summary
LCR Calculations
Long transformation protocol used for LCR reaction mixture.
Long Transformation
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