Haynes Lab:Notebook/Engineering PC-TFs/2012/11/06: Difference between revisions
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==Summary== | ==Summary== | ||
* <u>Gibson Assembly </u> | * <u>Gibson Assembly </u> | ||
* Check with PCR | |||
Assembly 1 contains PCR rxns 1,2, and 3. Assembly 2 contains PCR rxns 4,5. | Assembly 1 contains PCR rxns 1,2, and 3. Assembly 2 contains PCR rxns 4,5. | ||
There is no addition of vector this time. | There is no addition of vector this time. | ||
{|border="1" cellpadding="5" cellspacing="0 | {|border="1" cellpadding="5" cellspacing="0" | ||
|- | |- | ||
! | ! style="background:#efefef;" | | ||
! | ! style="background:#efefef;" | 1 | ||
! | ! style="background:#efefef;" | 2 | ||
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{|border="1" cellpadding="5" cellspacing="0 | {|border="1" cellpadding="5" cellspacing="0" | ||
|- | |- | ||
! scope="col" style="background:#efefef;" | <i>DNA fragments</i> | ! scope="col" style="background:#efefef;" | <i>DNA fragments</i> | ||
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|5.0 μL | |5.0 μL | ||
|} | |} | ||
<br><br><br><br> | |||
---- | |||
'''PCR Check''' | |||
# Product 1: outer-most primers for whole assembly | |||
# Product 1: Gibson primer pair for hPCD | |||
# Product 2: outer-most primers | |||
# Product 2: Gibson primer pair for hPCD | |||
<br> | <br> | ||
{|border="1" cellpadding="5" cellspacing="0" | |||
|- | |||
| <i>Reagent</i> || 1 || 2 || 3 || 4 | |||
|- | |||
| Gibson product || 0.5 μL || 0.5 || 0.5 || 0.5 | |||
|- | |||
| primer 1 || 1.0 || 1.0 || 1.0 || 1.0 | |||
|- | |||
| primer 2 || 1.0 || 1.0 || 1.0 || 1.0 | |||
|- | |||
| 2x GoTaq PCR mix || 14.5 || 14.5 || 14.5 || 14.5 | |||
|- | |||
| dH<sub>2</sub>O || 12.0 || 12.0 || 12.0 || 12.0 | |||
|} | |||
<br> | <br> | ||
<br> | <br> | ||
Program: | |||
* 95°C, 3 min. | |||
* [95°C, 30 sec; 57°C, 30 sec; 72°C, 30 sec] 30 cycles | |||
* 72°C, 3 min. | |||
* 4°C ∞ | |||
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Revision as of 16:55, 6 November 2012
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Summary
Assembly 1 contains PCR rxns 1,2, and 3. Assembly 2 contains PCR rxns 4,5. There is no addition of vector this time.
PCR Check
Program:
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