IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/07/01

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7/1/10

Meagan plated the cells transformed last night at 12:30 AM. We observed no growth on the plates this morning when we checked at 9:30 AM for both those plates and the PAL_pSB1A2 plate.

11:15 AM

Troubleshooting Gel

Ran ligations and digests on gels at 80V.

Lane Sample Amount
1. 1 Kb Ladder 2 ul
2. J23100 digest 5 ul sample, 1 ul dye
3. J23119 digest 5 ul sample, 1 ul dye
4. Gas Vesicles digest 5 ul sample, 1 ul dye
5. Tet Backbone digest 5 ul sample, 1 ul dye
6. J23100-Gas vesicle ligation 5 ul sample, 1 ul dye
7. J23119-Gas vesicle ligation 5 ul sample, 1 ul dye
8. pSBIT3 5 ul sample, 1 ul dye
9. PAL digest 5 ul sample, 1 ul dye
10. PAL ligation 5 ul sample, 1 ul dye

Image: UIUC_7.1_Digests,_Ligations,_etc(3-0.9et).jpg‎


Lane Sample Amount
1. 1 Kb Ladder 2 ul
2. pSB1A2 EcoRI/PstI 5 ul sample, 1 ul dye
3. pSB1T3 EcoRI/PstI 5 ul sample, 1 ul dye
4. pSB1C3 EcoRI/PstI 5 ul sample, 1 ul dye
5. pSB1K3 EcoRI/PstI 5 ul sample, 1 ul dye
6. pSB1A2 XbaI/SpeI/SAP 5 ul sample, 1 ul dye
7. pSB1T3 XbaI/SpeI/SAP 5 ul sample, 1 ul dye
8. pSB1C3 XbaI/SpeI/SAP 5 ul sample, 1 ul dye
9. pSB1K3 XbaI/SpeI/SAP 5 ul sample, 1 ul dye
10. pSB1K3 5 ul sample, 1 ul dye

Image:7.1_Plasmid_Digests_with_E,P_&_X,S.jpg‎

3:50 PM

Re-Ligating

J23100-Gas Vesicles-pSB1T3

H20 11 ul
J23100 2 ul
Gas Vesicles 2 ul
pSB1T3 2 ul
T4 buffer 2 ul
T4 Ligase 1 ul

J23119-gas vesicles-pSB1T3

H2O 11 ul
J23119 2 ul
gas vesicles 2 ul
pSB1T3 2 ul
T4 Buffer 2 ul
T4 Ligase 1 ul

Gas Vesicles-pSB1T3

H20 12 l
Gas Vesicles 2 ul
pSB1T3 2 ul
T4 buffer 2 ul
T4 Ligase 1 ul

incubate at room temperature for 10 minutes

inactivate at 80°C for 20 minutes

5:15 PM

Transformations

100 ul cells + 5 ul ligation product (J23100-Gas Vesicles-pSB1T3 or J23119-Gas Vesicles-pSB1T3). electroporate and add 1 mL LB, incubate at 37°C for 1 hour for recovery.

6:30 PM

Plating

Plated 20ul transformation product on 1/2 tetracycline plate, plated 200 ul on other half. Next day observed no growth.



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