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Sticky end preparation on plasmid and gene of interest
On Tuesday we did plasmid culture again with antibiotic Kanamycin because we made a mistake by using Ampicillin in LB in our last culture.We did Plasmid extraction by Gen jet plasmid mini prep kit. We also did the restriction digestion of PCR amplified accC gene by using enzymes PstI and XbaI and then purify with PCR purification kit.
On Thursday, we did agarose gel electrophorosis for purified Gene of interest and extracted plasmid DNA. We got good bands of expected size i.e 1350bp for our gene and approximately 3000bp for plasmid which is the expected size of plasmid DNA.Since our all plasmid samples showed similar band size we decided to mix all sample in one tube and make total volume 240ul. We also did restriction digestion and purification for plasmid DNA. We made a sticky ends by cutting it with enzyme PstI and SpaI to make it compatable with our gene of interest which is cut by PstI and XbaI. We, also did agarose gel electrophorosis for our digested plasmid. Unfortunately, we didn't saw any bright bands. However the bands were fragmented, the reason might be we made same mistake during purification process. We will go through details and discuss about the problem in our next lab.