User:Eduardo Vladimir Munoz/Notebook/UNAM Genomics Mexico 2011/2011/09/10

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High Stabilitiy Plasmid Development

Abstract

  • P1004 Band Isolation corroboration gel, and ligation concentration estimation gel.
  • Ligation of P1004 with B0014
  • Ligation of repC-PCR with B0015


Electrophoresis gel

1. Ladder 250bp
2. EMPTY
3. DD P1004 in pSB1A1 E,S
4. Band isolated P1004
5. DD KnR E,S
6. DD B0014 in pSB1AK3 E,X
7. DD B1007 in pSB1AK3 E,X
8. DD repC-PCR E,S
9. DD B0015 in pSB1AK3 st.2 E,X
10. pSB1C3 digestion with XhoI (provided by Fibo)


Ligations

P1004 and B0014 in pSB1AK3

Reaction 7:7 9:5
P1004 7μL 9μL
B0014 7μL 5μL
T4 DNA ligase 0.5μL 0.5μL
5x ligase buffer 4μL 4μL
H2O mQ 1.5μL 1.5μL
Total 20μL 20μL


repC-PCR and B0015 in pSB1AK3

Both DNA solutions were too concentrated, this is why I used more water here.

Reaction 6:4 7:4
repC-PCR 6μL 7μL
B0015 4μL 4μL
T4 DNA ligase 0.5μL 0.5μL
5x ligase buffer 4μL 4μL
H2O mQ 5.5μL 4.5μL
Total 20μL 20μL


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