GGA for BsmBI - Revision history

Macampbell at 15:36, 18 November 2014

2014-11-18T15:36:52Z

Macampbell at 15:35, 11 November 2014

2014-11-11T15:35:14Z

Macampbell at 19:01, 10 November 2014

2014-11-10T19:01:45Z

Macampbell: Created page with "We are trying to clone two gBlocks designed with sticky ends after digestion with BsmBI. '''Thermocycle Program''' * 20 minutes at 55 C (start with digestion cocktail below)..."

2014-11-10T18:54:15Z

Created page with "We are trying to clone two gBlocks designed with sticky ends after digestion with BsmBI. '''Thermocycle Program''' * 20 minutes at 55 C (start with digestion cocktail below)..."

New page

We are trying to clone two gBlocks designed with sticky ends after digestion with BsmBI.

'''Thermocycle Program'''
* 20 minutes at 55 C (start with digestion cocktail below)
* pause to put in 5 µL ligation mixture (see below)
* 5 minutes at 16 C
* (start loop below)
** 2 min 16 C
** 1 min 55 C
** (end loop) 20 cycles total
* 20 min 55 C
* hold at 21 C
<br>
<hr>
'''Digestion cocktail'''
** water 6.5 µL
** 25 ng vector cut with EcoRI and Spe (gel purified) (1.76 µL)
** 3x molar insert 1 (P1) 1 µL
** 3x molar insert 2 (P2) 1 µL
** 10x cutsmart buffer 1 µL
** BsmBI enzyme 0.5 µL
* final volume 10 µL
<br>
<br>
'''Ligation mixture'''
** water 3 µL
** 10X ligation buffer 1.5 µL
** ligase 0.5 µL

@@ Line 3: / Line 3: @@
 '''Thermocycle Program'''
 * 20 minutes at 55 C (start with digestion cocktail below)
 * 5 minutes at 16 C
-* (start loop below)
+* push pause when it reaches 16 C
 ** 2 min 16 C
 ** 1 min 55 C

@@ Line 9: / Line 9: @@
 ** 1 min 55 C
 ** (end loop) 20 cycles total
-* hold at 21 C
+* hold at 16 C
 <br>
 <hr>