9/7/2012
#
|
Biobrick
|
ng/µL
|
Size (bp)
|
1
|
hPCD : mCh : SP1AB
|
130.56
|
2520
|
2
|
hPCD : mCh : SP1A
|
266.898
|
1443
|
3
|
hPCD : mCh : SP1B
|
222.175
|
1224
|
4
|
hPCD : mCh : p65
|
189.63
|
1686
|
5
|
hPCD : mCh : VP64
|
66.561
|
1053
|
6
|
fshPCD : mCh : SP1AB
|
140.347
|
2520
|
7
|
fshPCD : mCh : SP1A
|
148.285
|
1443
|
8
|
fshPCD : mCh : SP1B
|
209.637
|
1743
|
9
|
fshPCD : mCh : p65
|
134.672
|
1686
|
10
|
fshPCD : mCh : VP64
|
263.567
|
1053
|
11
|
flyPCD : mCh : SP1A
|
43.068
|
1443
|
12
|
flyPCD : mCh : SP1B
|
35.838
|
1743
|
13
|
flyPCD : mCh : p65
|
83.886
|
1686
|
14
|
flyPCD : mCh : VP64
|
200.896
|
1053
|
Ligation
|
1
|
2
|
3
|
4
|
5
|
6
|
7
|
8
|
9
|
10
|
11
|
12
|
13
|
14
|
15
|
DNA Insert
|
.3 μL
|
.1 μL
|
.1 μL
|
.2 μL
|
.3 μL
|
.3 μL
|
.2 μL
|
.2 μL
|
.2 μL
|
.1 μL
|
.5 μL
|
.7 μL
|
.3 μL
|
.1 μL
|
0 μL
|
DNA Vector (hPCD)
|
.3 μL
|
.3 μL
|
.3 μL
|
.3 μL
|
.3 μL
|
.3 μL
|
.3 μL
|
.3 μL
|
.3 μL
|
.3 μL
|
.3 μL
|
.3 μL
|
.3 μL
|
.3 μL
|
.3 μL
|
T4 Ligase
|
1 μL
|
1 μL
|
1 μL
|
1 μL
|
1 μL
|
1 μL
|
1 μL
|
1 μL
|
1 μL
|
1 μL
|
1 μL
|
1 μL
|
1 μL
|
1 μL
|
1 μL
|
Ligation Buffer (2x)
|
5 μL
|
5 μL
|
5 μL
|
5 μL
|
5 μL
|
5 μL
|
5 μL
|
5 μL
|
5 μL
|
5 μL
|
5 μL
|
5 μL
|
5 μL
|
5 μL
|
5 μL
|
dH2O
|
3.4 μL
|
3.6 μL
|
3.6 μL
|
3.5 μL
|
3.4 μL
|
3.4 μL
|
3.5 μL
|
3.5 μL
|
3.5 μL
|
3.4 μL
|
3.2 μL
|
3.0 μL
|
3.4 μL
|
3.6 μL
|
3.7 μL
|
Total
|
10 μL
|
10 μL
|
10 μL
|
10 μL
|
10 μL
|
10 μL
|
10 μL
|
10 μL
|
10 μL
|
10 μL
|
10 μL
|
10 μL
|
10 μL
|
10 μL
|
10 μL
|
- In .5 ml tubes, pipette the ligation buffer first.
- Then pipette water, DNA insert, DNA vector, and T4 ligase in this order.
- Allowed them to incubate for 10 minutes.
- Retrieved ice bucket and allowed E.coli cells to thaw for five minutes.
- Followed the transformation process:
- Warmed 100 agar plates at 37°C.
- Thaw two fresh tubes of DH5α Turbo cells on ice.
- Add 30 μL of DH5α Turbo cells to DNA
- Incubated cells + DNA on ice for 5-10 minutes.
- Transfered cells + DNA onto agar plates
- Added 10-15 sterile glass beads to plates and shook plates.
- Discarded beads and incubated plates at 37°C°C overnight.
|