02/12/2015
Step 1: Cut backbone with EcoRI and PstI
- Make sure the small heat block is set to 37°C with the smallest tube size block inside
- Ryan aliquoted the backbone into two PCR tubes with 1ug of backbone in each tube and put them in your box
- Get from your box
- The two backbone tubes
- Fast digest buffer
- PstI
- EcoRI
- be sure to put the enzymes in the cold box when you bring them out of the freezer
- Add the following directly to the tube with the backbone:
Thing
|
vol in ul
|
1ug sample |
6
|
H2O |
10
|
10x buffer |
2
|
PstI |
1
|
EcoRI |
1
|
Total |
20
|
- Mix, spin, and incubate the two tubes at 37°C for 10 minutes
- Close all caps and return everything else to your box in the freezer
Step 2: Dephosphorylate cut ends
- Bring the rApid Alkaline Phosphatase from you box to the bench in the ice box
- Add 2ul of phosphatase to each digestion reaction.
- Mix, spin, and incubate at 37°C for 15-60 mins
- if you're short on time, you can do 15, if you have more time, I recommend a little longer for more complete dephosphorylation
- Close all caps and return everything else to your box in the freezer
Step 3: Purify cut backbone
- Combine the two digested and dephosphorylated samples into one column and elute in 20ul like you did today with the Sigma PCR purification kit
- Measure the concentration and put in your box in the freezer
Results
These are the results obtained after eluting the dephosphorylated and digested backbone twice. The first elution has a much higher concentration than the second elution, which is what we expected.
Sample Read#
|
260
|
280
|
260/280
|
ng/µL
|
1 (first elution) |
0.063 |
0.032 |
1.934 |
62.779
|
2 (second elution) |
0.006 |
0.002 |
2.81 |
6.212
|
|