840:153g:Projects/project25/2012/11/08

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Cloning of mreB from Caulobacter crescentus into E. coli Main project page
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Exp 6: Prepatory Digest, Purification, Electrophoresis. & Exp 7:Ligation of Insert and Vector

Tuesday:

We ran a gel of our digested vector (part 1 and 2) and digested gene segment with a 100bp plus DNA ladder and FastRuler Low Range DNA Ladder. We expected to see our Gene segment and vector samples at the correct length and the wells be very clean. The intensity of the bands, in comparison with the FastRuler Low Range DNA Ladder, will give us an estimated amount of DNA per microliter. This will let us figure out our insert:vector ratios for ligation, which will ideally be 2:1.



Well 1: 100 bp plus DNA Ladder. Well 2: 3μL of FastRuler. Well 3: Gene segment. Well 4: 10μL of FastRuler. Well 5: Vector Part 1. Well 6: 15μL of FastRuler. Well 7: Vector Part 2. The band lengths for the FastRuler are 1500, 850, 400, 200, 50. The FastRuler has 4ng/μL of DNA.

Our gene segment and vector came out at the correct length, and the wells were fairly clean. The intensity of our gene segment shows about 3-4 times brighter than the 1500 bp 15μL FastRuler in well 6, while vector Part 2 came out to be twice as bright. This gives us our ideal 2:1 (insert:vector) ratio for our ligation.

Thursday:

We set up our ligation reactions. We are running 3 tests with 3 controls. Test 1: 1:1 (insert:vector) ratio. Test 2: 1:5. Test 3: 5:1. The given numbers is the amount of μL for each reaction. The reactions will be kept at 4°C until Tuesday. After we finish ligations, we will perform Transformation into E.Coli. This is when we can see if our insert and vectors were combined and put into E.Coli. And hopefully see a change in cell shape!

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