# IGEM:MIT/2007/Notebook/2007-6-14

(Difference between revisions)
 Revision as of 09:53, 14 June 2007 (view source)← Previous diff Revision as of 09:57, 14 June 2007 (view source)Next diff → Line 21: Line 21: Start: 2 vials of competent bacteria (500 uL each)
Start: 2 vials of competent bacteria (500 uL each)
9 Transformations
9 Transformations
- *U V (2 uL) +
-                                                                *dV (5 uL)                                                     +                                                            Contents    Volume
-                                                                *dV + dI (5 uL)                                                +                                                            U V      (2 uL)
-                                                                *ddV (5 uL)                                                    +                                                            dV      (5 uL)
-                                                                *ddV + ddI (5 uL)                                              +                                                            dV + dI    (5 uL)
-                                                                *dV (10 uL)                                                    +                                                            ddV       (5 uL)
-                                                                *dV + dI (10 uL)                                               +                                                            ddV + ddI    (5 uL)
-                                                                *ddV (10 uL)                                                   +                                                            dV       (10 uL)
-                                                                *ddV + ddI (10 uL)

+ dV + dI     (10 uL) + ddV     (10 uL) + ddV + ddI     (10 uL) +

#Aliquot 5 uL and 10 uL of ligation reactions into eppendorfs #Aliquot 5 uL and 10 uL of ligation reactions into eppendorfs

## Revision as of 09:57, 14 June 2007

• Morning: Debbie
• Afternoon: Brian

## LAB WORK

• Make competent E. coli cells
• Transformation into competent cells
• Plate transformed cells

Depending on how fast cells grow......

• Night time
• Analyze plates
• innoculate 4ml LB/Amp

## Procedures

Regular cell membranes are not permeable to plasmids, so we much use ions (CaCl) to make the bacteria competent and able to accept foreign DNA.
Start: 2 vials of competent bacteria (500 uL each)
9 Transformations

```Contents    Volume
U V      (2 uL)
dV      (5 uL)
dV + dI    (5 uL)
ddV       (5 uL)
ddV + ddI    (5 uL)
dV       (10 uL)
dV + dI      (10 uL)
ddV      (10 uL)
ddV + ddI     (10 uL)
```

1. Aliquot 5 uL and 10 uL of ligation reactions into eppendorfs
• For the undigested vector, dilute 100x so we can put in around 2 ng (because optical density of undigested vector is 86.4 ng/uL)
2. Pipette 50 uL of competent DH5a E. coli bacteria into ligation reaction plasmid eppendorfs from above and mix by tapping gently
• Do not pipette up and down
3. Incubate on ice for 60 minutes
4. Heat shock the bacteria in a 37C water bath for 30 seconds