Gene Expression and Proteomics


Protocol for Ligation Reaction

1. Add the following: (be sure to add the dH2O and T4 Ligase 10x Buffer first; spin down if necessary.)

                                                     Ligation 1    Control
dH2O                                              8 uL          11 uL
T4 Ligase 10x Buffer                     2 uL          2 uL
Insert                                              3 uL             -
T4 Ligase                                        1 uL          1 uL
Vector-R.E.-CIP                              6 uL          6 uL
Total volume: 20 uL

2. Incubate the ligation mixture at room temperature for one hour.
3. Place on ice to cool down.

Protocol for Transformation Using Chemical Competent Cells

1. Obtain suitable antibiotic LB plates and warm up in 37°C incubator.
2. Warm up chemical competent dH5a cells on ice for 40 minutes. (500 uL of competent cells per tube.) Note: these competent cells are amp resistant, thus, we cannot use an amp resistant vector.
3. Obtain a new microtube, place on ice, and add 15 uL of ligation mixture.
4. Add 200-250 uL of competent cells to ligation mixture. Be sure to keep everything on ice.
5. Incubate on ice for one hour. Gently tap the microtube every 15 minutes during that hour.
6. Heat shock the mixture at 42°C for 60 seconds and then on ice for 60 seconds.
7. Add 1 mL of LB at room temperature to the mixture into the microtube and incubate at 37°C, 250 rpm for one hour.
8.Spread 200 uL of mixture on LB plate. (Add 200 uL of mixture to the LB plate; dip spreader into ethanol and place over open flame to sterilize for a few seconds; count to 100 to allow the spreader to cool down.)
8. Centrifuge the remaining mixture at level 7 for 10 seconds. Pour off approximately 600 uL of the supernatant. Spread the remaining on second LB plate.
10. After spreading, allow the plates to air dry by opening the lid about half way. Set timer for 10 minutes and check periodically.
11. Incubate overnight at 37°C up-side down.