Team:Uppsala-SwedenWeek12

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Latest revision as of 18:27, 26 October 2010

Week-12

Construction of I1 to I3

5 colonies were picked from each of those plates and sent to perform c-PCR. According to the band size on gel picture, 1 or 2 colonies of each set were selected to inoculate.

I1 I2

I2 I3

Selected colonies were inoculated overnight for getting enough material for doing plasmid extraction.Glycerol stocks were made before performing plasmid extraction. After extracting the plasmids, the concentration was measured. The plasmids were digested with the appropriate enzymes as per the flowchart and purified to remove the enzymes. The correct parts were ligated using a Tetracycline backbone.

Unfortunately the next day we did not get any colonies for all the 3 plates. It seems that the Tetracycline plates were not effective. As we were not sure about the effectiveness of the Tetracycline plates, we did the ligation and transformation again using Tetracycline backbone and also another replicate using Chloramphenicol backbone.

Colonies were obtained only for the Chloramphenicol backbone transformations. Hence, we decided to go ahead with using these colonies itself. However, our initial plan was upset and we would not end up with C3 backbone for the final constructs. At this time we felt the need to have more antibiotic resistance backbones to have more flexibility in our plan and overcome such problems. We were not able to obtain any colonies with Tetracycline plates until the end of the project.

@@ Line 2: / Line 2: @@
 == Week-12 ==
-== Construction of H1- G8 ==
+== Construction of I1 to I3 ==
 colonies were picked from each of those plates and sent to perform c-PCR. According to the band size on gel picture, 1 or 2 colonies of each set were selected to inoculate.
-[[Image:20100817 H1-H2-H8 c-PCR 20100814.jpg|500px|thumb|center|border|H1-H2-H8]]
+[[Image:20100819 I1 I2(2).jpg|500px|thumb|center|border|I1 I2]]
-From the above gel picture it was not very clear. Some fiddling around with the controls and we got a much better image (given below).
-[[Image:20100817 H1-H2-H8 c-PCR 20100814(2).jpg|500px|thumb|center|border|H1-H2-H8 with different illumination to identify the constructs]]
+[[Image:20100819 I2 I3(3).jpg|500px|thumb|center|border|I2 I3]]
-[[Image:20100817 H3--H4 c-PCR 20100814.jpg|500px|thumb|center|border|H3-H4]]
+Selected colonies were inoculated overnight for getting enough material for doing plasmid extraction.Glycerol stocks were made before performing plasmid extraction. After extracting the plasmids, the concentration was measured. The plasmids were digested with the appropriate enzymes as per the flowchart and purified to remove the enzymes. The correct parts were ligated using a Tetracycline backbone.
-[[Image:20100817 H5-H6 c-PCR 20100814(3).jpg|500px|thumb|center|border|H5-H6]]
+Unfortunately the next day we did not get any colonies for all the 3 plates. It seems that the Tetracycline plates were not effective. As we were not sure about the effectiveness of the Tetracycline plates, we did the ligation and transformation again using Tetracycline backbone and also another replicate using Chloramphenicol backbone.
-[[Image:20100817 H7-H8 c-PCR 20100814(3).jpg|500px|thumb|center|border|H7-H8]]
+Colonies were obtained only for the Chloramphenicol backbone transformations. Hence, we decided to go ahead with using these colonies itself. However, our initial plan was upset and we would not end up with C3 backbone for the final constructs. At this time we felt the need to have more antibiotic resistance backbones to have more flexibility in our plan and overcome such problems. We were not able to obtain any colonies with Tetracycline plates until the end of the project.
-The samples selected in Colony PCR were inoculated for obtaining material to do plasmid extraction. Overnight culture was used to make glycerol stocks and plasmids were extracted. The plasmid concentration was measured for estimating the amount of enzymes required for digestion and ligation.
-Plasmids were digested with proper enzymes bought from Sigma-Aldrich and run for gel to confirm the size. The plasmid parts where also digested for use in ligation for the next step.The correct set of parts was ligated using Ampicillin backbone via 3A assembly following the Flowchart as per the plan.