IGEM:UC Berkeley/2006/Plasmid Transformation

From OpenWetWare

< IGEM:UC Berkeley/2006(Difference between revisions)
Jump to: navigation, search
Current revision (14:50, 1 August 2006) (view source)
 
(4 intermediate revisions not shown.)
Line 1: Line 1:
-
'''KCM-competent cell heat shock'''<br>
+
'''KCM-competent cell heat shock (don't use this procedure for ligation products or cotransformations)'''<br>
  1. create cocktail on ice<br>
  1. create cocktail on ice<br>
-
   *20 uL TGI cells (for a cotransformation use 100uL of cells
+
   *20 uL TGI cells (for a cotransformation use 100uL of cells)<br>
-
0<br>
+
   *10 uL KCM buffer<br>
   *10 uL KCM buffer<br>
   *70 uL water<br>
   *70 uL water<br>
Line 12: Line 11:
-JL_06/09/06 <br>
-JL_06/09/06 <br>
<br>
<br>
-
'''Transformation of Ligation mix'''
+
'''Transformation of Ligation mix( use for ligation product transformations or cotransformations)'''
  1. Create cocktail on ice<br>
  1. Create cocktail on ice<br>
   *100 uL TGI cells<br>
   *100 uL TGI cells<br>
Line 26: Line 25:
<br>
<br>
'''Electro Shock Competence'''<br>
'''Electro Shock Competence'''<br>
-
  1. Set volts: 1.60 (slow thaw cells on ice) Use 2IT media or SOB media<br>
+
  1. Set volts: 1.60 (slow thaw cells on ice) Use 2YT media or SOB media<br>
-
  2. Put 50 mL of cells into clean eppendorf and limit bubbles. Add 1 uL of ligation mixture and stir to mix.<br>
+
  2. Put 50 uL of cells into clean eppendorf and limit bubbles. Add 1 uL of ligation mixture and stir to mix.<br>
  3. Pipette into electro cuvette with its cap off. Tap down to bottom, dry the tube<br>
  3. Pipette into electro cuvette with its cap off. Tap down to bottom, dry the tube<br>
  4. Put into holds, well seated in back <br>
  4. Put into holds, well seated in back <br>
-
  5. After beep, rescue with 500 mL(have ready) of media. Transfer all volume back to eppendorf, no longer on ice.<br>
+
  5. After beep, rescue with 500 uL(have ready) of media. Transfer all volume back to eppendorf, no longer on ice.<br>
-SIL_06/19/06 <br>
-SIL_06/19/06 <br>
<br>
<br>
 +
 +
 +
==5X KCM==
 +
#0.5M KCl
 +
#0.15M CaCl2
 +
#0.25M MgCl2

Current revision

KCM-competent cell heat shock (don't use this procedure for ligation products or cotransformations)

1. create cocktail on ice
*20 uL TGI cells (for a cotransformation use 100uL of cells)
*10 uL KCM buffer
*70 uL water
*1 uL plasmid (for cotransformation use 2uL of each plasmid)
2. heat shock for 1.5 min at 42 C
3. incubate on ice for 5 min.
3.5 If antibiotic marker is anything but Amp then rescue cells in ~100uL (or about equal volume) LB for one hour at 37C.
4. plate

-JL_06/09/06

Transformation of Ligation mix( use for ligation product transformations or cotransformations)

1. Create cocktail on ice
*100 uL TGI cells
*15 uL KCM buffer
*25 uL water
*10 uL plasmid
2. Incubate on ice for 10 min.
3. Heat shock for 1.5 min at 42 C
4. Rescue: add 150uL of SOB media
5. Incubate in 37 C shaker for 1 hour
6. Plate

-JL_06/14/06

Electro Shock Competence

1. Set volts: 1.60 (slow thaw cells on ice) Use 2YT media or SOB media
2. Put 50 uL of cells into clean eppendorf and limit bubbles. Add 1 uL of ligation mixture and stir to mix.
3. Pipette into electro cuvette with its cap off. Tap down to bottom, dry the tube
4. Put into holds, well seated in back
5. After beep, rescue with 500 uL(have ready) of media. Transfer all volume back to eppendorf, no longer on ice.

-SIL_06/19/06


5X KCM

  1. 0.5M KCl
  2. 0.15M CaCl2
  3. 0.25M MgCl2
Personal tools