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Engineering PC-TFs
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3/27/2013 1. Kozak,V0120 + BL01
2. Kozak,V0120 + BL05
3. Kozak,V0120 + BL09
4. Kozak,V0120 + BL12
#
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
µL of insert = (length insert/lengthvector *2*25 ng vector/concentration insert)
1
2
3
4
5
DNA Insert
1.4
1.8
2.0
2.5
--
DNA Vector (Kozak)
0.6
0.6
0.6
0.6
0.6
T4 Ligase
1
1
1
1
1
Lign Buffer (2x)
5
5
5
5
5
dH2O
2.0
1.6
1.4
.9
3.7
10 µL
10 µL
10 µL
10 µL
10 µL
In a .5 mL tube, pipette the ligation buffer first.
Then water, DNA insert, vector, and T4 ligase.
Once completed, allow them to incubate for 10 minutes.
Meanwhile, get ice bucket and allow competent E. coli cells to thaw for five minutes.
Then follow transformation process: Transformation Process
Warm five 100 μg/mL Amp agar plates at 37 °C
Thaw fresh tube of DH5α Turbo cells on ice
Add 30 μL of DH5α Turbo cells to DNA + dH2O
Include #5 tube, water only, no DNA (negative control)
Incubate cells + DNA on ice for 10 min.
Label pre-warmed plates
Transfer cells + DNA onto agar
Add 10 - 15 sterile glass beads, shake, discard beads
Incubate plates at 37 °C overnight Ligation attempt 3/27/2013
No colonies on any of the plates. Try again.
Engineering PC-TFs
