3/18/22
Cutting inserts to be placed in mammalian vector
A) Get biobricks and enzymes from freezer.
SpeI, PstI, XbaI
The following list of parts are:
Stock Digest
B)Follow digest procedure:
DNA 20.0 µL
Enzyme 1 1.0 µL
Enzyme 2 1.0 µL
10x buffer 3.0 µL
dH2O 5.0 uL
= 30.0 uL
|
Digest Order:
Biobrick Restriction Enzymes
- 1. BL01 XbaI/PstI
- 2. BL05 XbaI/PstI
- 3. BL09 XbaI/PstI
- 4 BL12 XbaI/PstI
Follow steps for gel electrophoresis.
- Orient top notch of gel electrophoresis apparatus. Place gel tray into larger tray.
- Fill gel flask with up to 60 ml of TA buffer.
- Create 1% gel by putting .6 grams of agarose into flask.
- Microwave agarose solution for 40 seconds
- Using hot gloves, grab flask out of microwave and swirl flask vigorously. Place back in microwave for 40 more seconds.
- When flask is taken out of microwave, make sure that the agarose is completed dissolved.
- Set up wells on gel electrophoresis apparatus. The mold for the first digest is B-10. Use the thick "teeth" and slip in the notches.
- Add EtBr (ethidium bromide) to the agarose gel. There should be 1 microliter of EtBr per 10ml of agarose gel. Therefore, 6 µL.
- Pour gel into tray.
- Wash the agarose gel flask.
- Run gel for 45 min at 100 V. Check gel under UV light.
Reagent
|
Volume
|
30 μL/lane, 1% agarose; Ladder
|
DNA (plasmid) |
15.0 μL
|
XbaI |
1.0 μL
|
PstI |
1.0 μL
|
Green 10x FastDigest buffer |
1.0 μL
|
dH2O |
10 μL
|
|
30 μL --> 37°C/ ~10 min.
|
#
|
Biobrick
|
ng/µL
|
1
|
hPCD : mCh : SP1AB (BL01)
|
18.834
|
2
|
fshPCD : mCh : SP1AB (BL05)
|
14.471
|
3
|
flyPCD : mCh : SP1A (BL09)
|
7.302
|
4
|
hPCD : BL01 (BL12)
|
10.84
|
|