Haynes Lab:Notebook/Engineering PC-TFs/2014/08/04: Difference between revisions
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(Autocreate 2014/08/04 Entry for Haynes_Lab:Notebook/Engineering_PC-TFs) |
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==Summary== | ==Summary== | ||
* | '''Ligation''' | ||
Calculations resulted in the following: | |||
{| class="wikitable" width=400px | |||
| || <u>Ligation</u> || <u>Negative Control</u> | |||
|- | |||
| Insert DNA (BL01, BL05, BL09 respectively) || 12μL || '''none''' | |||
|- | |||
| Vector DNA (50 ng) || 1μL || same | |||
|- | |||
| 10x Biolabs T4 Ligase Buffer || 2.0 μl || same | |||
|- | |||
| Biolabs T4 Ligase || 1.0 μl || same | |||
|- | |||
| dH<sub>2</sub>O || 4.0μL || 16.0μL | |||
|- | |||
| || 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. | |||
*Six 1.5mL tubes were setup and labeled; 1 (BL01), 5 (BL05), 9 (BL09), CMV9 (CMV/MV9, ligation benchmark), rhlI (pos control), Neg (negative control). | |||
*60μL of thawed cells were mixed and then transferred into each of these six empty tubes. | |||
*The ligation products were then transferred into their respective tubes and flicked to mix. Set back on ice. | |||
*The RhlI plasmid 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> | |||
---- | |||
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Revision as of 12:10, 5 August 2014
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SummaryLigation Calculations resulted in the following:
Long Transformation
|