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.
|
Engineering PC-TFs
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