Adam Crego's Laboratory Notebook

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(02/06/08 '''Transformation of DNA''')
(02/06/08 '''Transformation of DNA''')
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====Transformation of Recombinant DNA====
====Transformation of Recombinant DNA====
#) Obtain specific plate from refrigerator.  A number and a letter.  ex. 3J
#) Obtain specific plate from refrigerator.  A number and a letter.  ex. 3J
-
# Set to 8 or 9 microL on pipette
+
#) Set to 8 or 9 microL on pipette
-
# Inject distilled water into the well (pipette up and down a few times) and mix.
+
#) Inject distilled water into the well (pipette up and down a few times) and mix.
-
# Water should be orange from DNA.
+
#) Water should be orange from DNA.
Line 22: Line 22:
====Transformations:====
====Transformations:====
-
1.) If using DNA from Registry, dilute with 9 microL of H20.
+
#) If using DNA from Registry, dilute with 9 microL of H20.
-
2.) Label 2 tubes w/ (part/date/name)
+
#) Label 2 tubes w/ (part/date/name)
-
3.) Add 100 microL competent cells (strain of E. coli : JM109, company Promega, catalog #L2001) in each tube
+
#) Add 100 microL competent cells (strain of E. coli : JM109, company Promega, catalog #L2001) in each tube
-
4.) Add 4 microL of Registry DNA into only one tube
+
#) Add 4 microL of Registry DNA into only one tube
-
5.) Place in ice bath for 30 minutes
+
#) Place in ice bath for 30 minutes
-
6.) Heat shock at 42 degrees C for 45-60 seconds
+
#) Heat shock at 42 degrees C for 45-60 seconds
-
7.) Place in ice bath for 2 minutes
+
#) Place in ice bath for 2 minutes
-
8.) Add 900 microL ice cold antibiotic free LB to cells
+
#) Add 900 microL ice cold antibiotic free LB to cells
-
9.) Incubate at 37 degrees C for 90 minutes in a shaking incubator
+
#) Incubate at 37 degrees C for 90 minutes in a shaking incubator
-
10.) Dilute as desired, make necessary controls, and plate cells (200 microL per plate)
+
#) Dilute as desired, make necessary controls, and plate cells (200 microL per plate)
-
11.) Incubate upside down at 37 degrees C for 18-24 hours, then refrigerate
+
#) Incubate upside down at 37 degrees C for 18-24 hours, then refrigerate
3 Plates:
3 Plates:
-
1.) Control: 100 microL LB/cell
+
#) Control: 100 microL LB/cell
-
2.) 2 test plates
+
#) 2 test plates
1:1 100 micro LB/cells
1:1 100 micro LB/cells
1:100 1 microL cells to 99 microL H20
1:100 1 microL cells to 99 microL H20

Revision as of 17:42, 6 February 2008

02/06/08 Transformation of DNA

Name: control, Part BBa_J45219 and BBa_R0011R


Group 1:

Parts: (B0032) --> ATF1 (J45014) --> (B0010) --> (B0012) Library Well Plate Plasmid Cell iGEM 2007 3J iGEM 2007 Parts Kit Plate 3 pSB1AK3 V1009 Available

Transformation of Recombinant DNA

  1. ) Obtain specific plate from refrigerator. A number and a letter. ex. 3J
  2. ) Set to 8 or 9 microL on pipette
  3. ) Inject distilled water into the well (pipette up and down a few times) and mix.
  4. ) Water should be orange from DNA.


        • Only need 4 microL for a transformation (part/date/initials on tube)

• Making lots of DNA - Amplifying using PCR • Save some cells (DNA) in freezer at -80 degrees C

Transformations:

  1. ) If using DNA from Registry, dilute with 9 microL of H20.
  2. ) Label 2 tubes w/ (part/date/name)
  3. ) Add 100 microL competent cells (strain of E. coli : JM109, company Promega, catalog #L2001) in each tube
  4. ) Add 4 microL of Registry DNA into only one tube
  5. ) Place in ice bath for 30 minutes
  6. ) Heat shock at 42 degrees C for 45-60 seconds
  7. ) Place in ice bath for 2 minutes
  8. ) Add 900 microL ice cold antibiotic free LB to cells
  9. ) Incubate at 37 degrees C for 90 minutes in a shaking incubator
  10. ) Dilute as desired, make necessary controls, and plate cells (200 microL per plate)
  11. ) Incubate upside down at 37 degrees C for 18-24 hours, then refrigerate

3 Plates:

  1. ) Control: 100 microL LB/cell
  2. ) 2 test plates

1:1 100 micro LB/cells 1:100 1 microL cells to 99 microL H20

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