Haynes Lab:Notebook/Engineering PC-TFs/2013/10/19: Difference between revisions
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==October 19, 2013== | ==October 19, 2013== | ||
''Ligation of CMV + MV9'' | ''Ligation of CMV + MV9'' | ||
{| {{table}} | |||
| align="center" style="background:#efefef;"| '''Part''' | |||
| align="center" style="background:#efefef;"|'''Concentration''' | |||
|- | |||
| CMV (insert)||15.535 ng/μL | |||
|- | |||
| MV9 (vector)||20.838 ng/μL | |||
|- | |||
|} | |||
<span style="font-size:95%">''Calculations'' | |||
<span style="font-size:95%">ng insert = bp insert/bp vector * 2 * 50 <br> | |||
ng insert = 588/4611 * 2 *50 = 12.75211 ng | |||
<span style="font-size:95%">μL insert = ng insert/concentration insert<br> | |||
μL insert = 12.75211/15.535 = .82μL | |||
<span style="font-size:95%">μL vector = 50 ng/concentration vector<br> | |||
μL vector = 50/20.838 = 2.4 μL | |||
{| {{table}} | {| {{table}} | ||
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| align="center" style="background:#efefef;"|'''Control''' | | align="center" style="background:#efefef;"|'''Control''' | ||
|- | |- | ||
| DNA Insert|| | | DNA Insert||0.8||0 | ||
|- | |- | ||
| DNA Vector ( | | DNA Vector (MV9)||2.4||2.4 | ||
|- | |- | ||
| T4 Ligase||1||1 | | T4 Ligase||1||1 | ||
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| Lign Buffer (2x)||5||5 | | Lign Buffer (2x)||5||5 | ||
|- | |- | ||
| | | dH<sub>2</sub>O ||0.8||1.6 | ||
|- | |- | ||
| '''Total'''||10 µL||10 µL | | '''Total'''||10 µL||10 µL | ||
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*Warm 100 μg/mL Amp agar plate at 37°C | *Warm 100 μg/mL Amp agar plate at 37°C | ||
*Thaw fresh tube of DH5α Turbo cells on ice | *Thaw fresh tube of DH5α Turbo cells on ice | ||
*Add 30 μL of DH5α Turbo Cells to DNA + dH<sub>2</sub>O | *Add 30 μL of DH5α Turbo Cells to DNA + dH<sub>2</sub>O. | ||
** This was also done for transformation of part KAH126 | |||
*Incubate cells + DNA on ice for 5 minutes. | *Incubate cells + DNA on ice for 5 minutes. | ||
*Label pre-warmed plate. | *Label pre-warmed plate. | ||
*Transfer cells + DNA onto agar. | *Transfer cells + DNA onto agar. | ||
*Add 8-10 glass beads, shake, then discard beads. | *Add 8-10 glass beads, shake, then discard beads. | ||
Incubate plates at 37°C overnight and retrieve next day. | *Incubate plates at 37°C overnight and retrieve next day. | ||
Revision as of 14:20, 20 October 2013
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October 19, 2013Ligation of CMV + MV9
Calculations ng insert = bp insert/bp vector * 2 * 50 μL insert = ng insert/concentration insert μL vector = 50 ng/concentration vector
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