840:153g:Projects/project24/2012/11/15

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(Autocreate 2012/11/15 Entry for 840:153g:Projects/project24)
Current revision (16:49, 29 November 2012) (view source)
(Sticky end preparation on plasmid and gene of interest)
 
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==PCR and Gel electrophorosis fro extracted DNA sample==
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==Sticky end preparation on plasmid and gene of interest==
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[[category:OWWLabNotebookV1]]
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On Tuesday, we ran DNA 3 sample at gel again because in last Agarose gel electrophorosis we didn't get band for DNA 3 however, the same sample had good ECORI digested band. We found that DNA 3 had similar intensity as DNA 2 and 5 so we mixed all three samples. We also,did PCR for our extracted DNA sample. This time we used non extended primers and run the samples at three different temperatures ( 65,55 and 50 degree centigrade). We also, tested for other group's DNA sample. To check if our primer is specific to our gene of interest or  not.
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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.
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On Thursday, we did Agarose gel electrophorosis for our PCR product. This time we used 3ul marker. The result seems to be satisfactory this time because, we saw that our DNA was amplified with non- extended primers and also, it was specific to our DNA because there was no amplification on DNA of other groups. When we compared the band size with 100bp Marker, we found that the size of DNA band was around 1350bp which is the size of our gene of interest. So, we decided to do PCR of our 3 samples using extended primers in our next lab.
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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 http://openwetware.org/wiki/Image:PIC_11.jpg. 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  Unfortunately, we didn't saw any bright bands http://openwetware.org/wiki/Image:PIC_12.jpg.  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.

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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 http://openwetware.org/wiki/Image:PIC_11.jpg. 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 Unfortunately, we didn't saw any bright bands http://openwetware.org/wiki/Image:PIC_12.jpg. 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.

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