Haynes Lab:Notebook/Engineering PC-TFs/2012/09/05

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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.



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