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What we did during five weeks:
1-Cloned GFP into TOPO
2-transformed PeGFPTOPO into E.Coli
3-counted CFU’s
4-Pick three colonies and Grew CFU’s in liquid broth and extracted the plasmid
5-Ran plasmid in Gel Electrophoresis
6- set up digest
7-Run digest on Gel Electrophoresis
8-Analyse Gel.
Title: should be specific ( Cloning of GFP into PCR TOPO).
-Aim: include the following: *demonstrate if we did create the plasmid?
*If we did get GFP in TOPO?
and confirm that we got GFP in TOPO?
-Introduction: we have to talk about :
Cloning, GFP in TOPO and restriction analysis.
-Result: we have to count the colonies in the + control and in the sample, we have got 59 CFU’S in the + control and no colonies in the sample. As well as that, Run Gel to look if we did got DNA load or not according to image 1.Use the gel image 2 that we have attach to analyse it ( determine the molecular size of each band and determine If the digestion DNA in each lane are cut, (while due to our result there is no cut).
-Discussion: Discus did we got colonies? ( + control work means translation work, and no colonies in the sample means the ligation reaction doesn’t work) , Did we got DNA ( from image 1) , and according to image 2 (why there are no cut in lane 2 and 3 ,and lane 4 is degraded?) what are the expected size product? (the size of the plasmid is 4673bp which digested with PGFP TOPO=4673bp, Pst1 = 4673bp and Ecor1=3938bp) and what size did we got from the image 2? why nothing is cut? what went wrong?( may the Gel not melt properly or let the Gel cool for too long).
-Conclusion: reflection of the aim.