Haynes Lab:Notebook/Engineering PC-TFs/2014/11/10: Difference between revisions
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==Summary== | ==Summary== | ||
* | *LCR | ||
*Long Transformation | |||
---- | |||
<br> | |||
'''LCR'''<br> | |||
Oligo bridges initially 30nm pellet. | |||
Diluted down to 30nM in 25μL reaction volume (Added 300μL H<sub>2</sub>O to IDT tube with pelleted DNA to yield 100μM, added 3μL of that dilution to 97μL H<sub>2</sub>O to yield 3μM, added 5μL of that dilution to 45μL H<sub>2</sub>O to yield 300nM, added 2.5μL of this dilution to the 25μL reaction volume for LCR).<br> | |||
<br> | |||
Diluted insert and vector down to 3nM using MW and known concentration.<br> | |||
<br> | |||
LCR Calculations | |||
* 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 BL01&5 = (2520bp/5197bp)*1*50ng vector = 24.24ng BL01&9 | |||
* Volume BL01&5 = 24.24ng*(1μL/10ng) = 2.424μL | |||
* Volume CMV/MV9 = 50ng*(1μL/33ng) = 1.28μL | |||
* 2:1 vector to insert use 2.56μL vector. | |||
{| class="wikitable" width=400px align="left" | |||
| || BL01|| BL05 | |||
|- | |||
| Insert DNA || 2.5μL || 2.5μL | |||
|- | |||
| XbaI Cut and Cleaned Vector DNA (CMV/MV9) || 1.3μL || 2.6μL | |||
|- | |||
| Oligo Bridge1 || 2.5μL || 2.5μL | |||
|- | |||
| Olido Bridge2 || 2.5μL || 2.5μL | |||
|- | |||
| 10X Ampligase Buffer || 2.5 μl || 2.5μL | |||
|- | |||
| Ampligase || 0.5μl || 0.5μL | |||
|- | |||
| Betaine || 0μL || 0μL | |||
|- | |||
| DMSO || 0μL || 0μL | |||
|- | |||
| dH<sub>2</sub>O || 13.2μL || 11.9μL | |||
|- | |||
| Total || 25.0 μL || 25μL | |||
|- | |||
| colspan="3" | Mix the reaction(s) thoroughly by flicking the tube.<br> Placed in thermocycler on LCR setting. | |||
|} | |||
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Long transformation protocol used for LCR reaction mixture.<br> | |||
<br> | |||
'''Ligation''' | |||
Calculations resulted in the following: | |||
{| class="wikitable" width=400px | |||
| || <u>Ligation</u> || <u>Negative Control</u> | |||
|- | |||
| Insert DNA (BL01, BL05 respectively) || 2.5 μL || '''none''' | |||
|- | |||
| Vector DNA (50 ng) || 1.3 μL || same | |||
|- | |||
| 10x Biolabs T4 Ligase Buffer || 2.0 μl || same | |||
|- | |||
| Biolabs T4 Ligase || 1.0 μl || same | |||
|- | |||
| dH<sub>2</sub>O || 13.2 μL || 15.7 μL | |||
|- | |||
| || 20.0 μL total || same | |||
|- | |||
| colspan="3" | | |||
|} | |||
<br> | |||
Mix the reaction(s) thoroughly by flicking the tube.<br> | |||
Incubate at room temperature for 45 minutes. | |||
<br> | |||
---- | |||
'''Long Transformation''' | |||
<br> | |||
Using the set up ligation products from above. | |||
*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 tube. | |||
*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 were heat shocked on the heat block at 42°C for 30 seconds and then placed back on ice for 2 minutes. | |||
*900μL LB broth (no antibiotic) was pipetted into each of the three tubes. | |||
*Tubes were taped into an empty plate. The plate was taped to the shaker within the incubator. | |||
*Incubated on shaker for 1 hour at 37°C and 240rpm. | |||
*Centrifuged for 1.5 minutes at 8 x g. | |||
*500μL media removed from each tube. | |||
*Resuspended cell pellet in remaining 400μL media. | |||
*300μL cells transferred onto respective plate. | |||
*Sterile glass beads used to spread cells onto plate. | |||
*Placed in the incubator for overnight growth. | |||
<br> | |||
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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