User:Eduardo Vladimir Munoz/Notebook/UNAM Genomics Mexico 2011/2011/09/08

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High Stabilitiy Plasmid Development

Abstract

  • Double Digestions of the plasmids isolated by Fibo yesterday (these).
  • Digestions corroboration gel
  1. False positive detected in the ligation of repC and pSB1AK3

Double Digestions

repC putative ligation

Reactive Digestion Double Dig.
Putative ligation plasmid 5μL 10μL
BSA 10x 1μL 2μL
Buffer 2 1μL 2μL
EcoRI-HF 1μL 1μL
SpeI 0 1μL
H2O mQ 2μL 6μL
Total 10μL 20μL

The double digestion must run farther than the simple digestion, and show another band.


B0015 in pSB1AK3

Reactive Stock 2 Stock 3
B0015 in pSB1AK3 22μL 22μL
BSA 10x 3μL 3μL
Buffer 4 3μL 3μL
EcoRI-HF 1μL 1μL
XbaI 1μL 1μL
H2O mQ 0 0
Total 30μL 30μL

Two more plasmid stocks (1 and 4) were extracted yet not digested.

P1004 in pSB1A1

Reactive Volume
P1004 in pSB1A1 22μL
BSA 10x 3μL
Buffer 2 3μL
EcoRI-HF 1μL
SpeI 1μL
H2O mQ 0
Total 30μL


Gel

Gel ran 40min at 120V, laded with 2μL of dye and 2μL of sample per lane:

Lane Sample
1. Ladder 500bp
2. Putative ligation of repC in pSB1AK3
3. EcoRI-HF linearized put. repC in pSB1AK3
4. DD E,S put. repC in pSB1AK3
5. B0015 in pSB1AK3 stock 2
6. DD B0015 E,X stock 2
7. B0015 in pSB1AK3 stock 3
8. DD B0015 E,X stock 3
9. P1004 in pSB1A1 stock 1
10. DD P1004 E,S
11. S1 (Dan)
12. S2 (Dan)

PCR Double Digestions

PCR repC 15μL PCR KnR 14μL
BSA 10x 2μL BSA 10x 2μL
Buffer 2 2μL Buffer 2 2μL
EcoRI-HF 0.5μL EcoRI-HF 0.5μL
SpeI 0.5μL SpeI 0.5μL
H2O mQ 0μL H2O mQ 1μL
Total 20μL Total 20μL



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