Team:TzuChiU Formosa/Result

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Latest revision as of 03:27, 22 October 2009

Result

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Figure.1(a.)pSB1A3 cloning PCR gel image. start with left to right part:(1.)100 bps marker,(2.)OmpC promoter,(3.)OmpC promoter,(4.)Negative control。(b.)the three red circles are single colony that have been transformed OmpC promoter containing plasmid。(c.)OmpC plasmid gel image。start with left to right part：(1.)100 bps marker,(2.)pSB1A3 plasmid,(3.)pSB1A3 plasmid.

　　Our PCR show that pSBA1B3 contained OmpC promoter, and the size was about 108 base pairs. From Fig.1-a Show that band was placed at 108 base pairs, which confirmed that pSB1A3 contained OmpC promoter. From Fig1-b we can see the growing of single colony. Fig1-c shows plasmid was at 2.75 Kb, so it was confirmed that pSB1A3 had been successfully transformed into DH5-α competent cell.

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Figure.2(a.)Aequorin-GFP sequence after purify gel image。Left:(1.)100 bps marker, (2.)Aequorin-GFP, (b.)After TA cloning transformation into DH5-α competent cell and run the blue white selection.(c.)Aequorin-GFP cloning PCR gel image. Start with left to right:(1.)100 bps marker,(2.3.)Aequorin-GFP.

　　The purify and concentration are unknown,so we run the purification to increase. Fig.2-a Show that template was placed at 2000 baes pairs. And took Aequorin-GFP to the TA cloning, let plasmisd could ligate to pGEM-T-easy plasmid. After transformation into DH5-α competent cell, then spirting the plate contain Ampicilin, 100ul IPTG, 50 ul X-gal. Fig2-b: Show that the blue white selection was white single colony. Picked two closing size single colony to amplify, and run colony PCR show that Aequorin-GFP size was about 2000 baes pairs. Fig.2-c Show that the sequence was at 2000 base pairs.

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Figure.3(a.)pSB1A3 digestion gel image. Start with left to right:(1.)100bs marker, (2.)Uncut pSB1A3, (2.)Uncut pSB1A3, (3.)Cut pSB1A3; (b.) (1.)100bps marker, (2.)Dephosphorelation of Pst1 cutting site in pSB1A3.

　　When we confirm the PSB1A3 that contain OmpC promoter. And we amplify it, used Pst1 to cutting off the sequences and run the gel electrophoresis. Due to the cutting form plasmid will change circular to linear form caused molecular weight increasing so the gel electrophoresis speed will slow down and stop at the upper strata show at the Fig.3-a.

　　In order to improve the successful rate of putting insert into the vector we treated, Pst1 digested pSB1A3 with calf Intestinal Alkaline Phosphatase (CIP) to remove the phosphate group thus reducing the chance of self ligation

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 ==Result==
-<gallery>
+.
+<gallery widths=250px heights=250px>
 Image:Result_f1a.jpg|a
 Image:Result_f1b.jpg|b
 Image:Result_f1c.jpg|c
 </gallery>
-'''Figure.1 (a.) SB1A3 cloning PCR gel image. start with left to right part:(M)100b.p. marker, (PS)PSB1A3,(PS)PSB1A3,(N)Negative control. (b.) the plate is PSB1A3 transformation into DH5α’s single colony.(c.)ompC plasmid gel image. start with left to right part:(M) 100b.p. marker, (C)ompC,(C)ompC.'''
+'''Figure.1(a.)pSB1A3 cloning PCR gel image. start with left to right part:(1.)100 bps marker,(2.)OmpC promoter,(3.)OmpC promoter,(4.)Negative control。(b.)the three red circles are single colony that have been transformed OmpC promoter containing plasmid。(c.)OmpC plasmid gel image。start with left to right part：(1.)100 bps marker,(2.)pSB1A3 plasmid,(3.)pSB1A3 plasmid.'''
-According to cloning PCR experiment we could make sure that PSB1A3 contain OmpC, and come out that OmpC size was about 108 base pair ; Fig.1-a show that template was at 108 bp, so it confirm PSB1A3 really contain OmpC promoter. After transform PSB1A3 into DH5α, culturing and spirting; Fig1-b: Grows single colony. Picking two closing size single colony, after culture in 37℃ and amplified, drawing out PSB1A3 plasmid and run gel electrophoresis to check did transformation has been success. The PSB1A3 size was about 2.75kb; Fig1-c: show plasmid was at 2.75kb, so it confirm PSB1A3 had successful transform into DH5α.
+　　Our PCR show that pSBA1B3 contained OmpC promoter, and the size was about 108 base pairs. From Fig.1-a Show that band was placed at 108 base pairs, which confirmed that pSB1A3 contained OmpC promoter. From Fig1-b we can see the growing of single colony. Fig1-c shows plasmid was at 2.75 Kb, so it was confirmed that pSB1A3 had been successfully transformed into DH5-α competent cell.
+.
-<gallery>
+<gallery widths=250px heights=250px>
 Image:Result_f2a.jpg|a
 Image:Result_f2b.jpg|b
 Image:Result_f2c.jpg|c
 </gallery>
-'''Figure.2(a.)Aqueorin-GFP sequence after purify gel image. Left:(M)100b.p. marker,(AG)Aqueorin-GFP.(b.)After TA cloning transformation into DH5α，spirt and run the blue white selection.(c.)Aequorin-GFP cloning PCR gel image. Start with left to right:(M)100b.p.marker, the other(A-G)Aqueorin-GFP.'''
+'''Figure.2(a.)Aequorin-GFP sequence after purify gel image。Left:(1.)100 bps marker, (2.)Aequorin-GFP, (b.)After TA cloning transformation into DH5-α competent cell and run the blue white selection.(c.)Aequorin-GFP cloning PCR gel image. Start with left to right:(1.)100 bps marker,(2.3.)Aequorin-GFP.
+'''
-Because of Aqueorin-GFP didn't know the purify and concentration;after PCR then we run the purify step to increasing the purify. The Aqueorin-GFP size was about 2000bp;Fig.2-a show that template was placed at 2000 bp, so it confirm that suecssfully purify the Aqueorin-GFP. And take the successful purify Aqueorin-GFP to run the TA cloning, let Aqueorin-GFP could ligate to Pgem-T-easy plasmid. After transforma into DH5α, then spirting the plate contain Ampicilin, 100ul IPTG, 50ul X-gal. As the result of blue white selection was white single colony which was we expect.Fig2-b: Show that the white single colony was the successful form. And we pick from the two closing size single colony after put at the 37oc and amplify, then run cloning PCR show that Aquorin-GFP size was about 2000bp. Fig.2-c show that the sequence was placed at 2000bp，so it confirm Aqueorin-GFP had been successfully ligate to Pgem-T-easy plasmid.
+　　The purify and concentration are unknown,so we run the purification to increase. Fig.2-a Show that template was placed at 2000 baes pairs. And took Aequorin-GFP to the TA cloning, let plasmisd could ligate to pGEM-T-easy plasmid. After transformation into DH5-α competent cell, then spirting the plate contain Ampicilin, 100ul IPTG, 50 ul X-gal. Fig2-b: Show that the blue white selection was white single colony. Picked two closing size single colony to amplify, and run colony PCR show that Aequorin-GFP size was about 2000 baes pairs. Fig.2-c Show that the sequence was at 2000 base pairs.
+.
-<gallery>
+<gallery widths=250px heights=250px>
-Image:Result_f3a.jpg|a
+Image:Result_f3a.jpg|3a
+Image:Result_f3b.JPG|3b
 </gallery>
-'''Figure.3(a.)PSB1A3 digestion gel image。Start with left to right:(M)100b.p.marker,(None)None cut PSB1A3,(None)None cut PSB1A3,(cut)cut PSB1A3.'''
+'''Figure.3(a.)pSB1A3 digestion gel image. Start with left to right:(1.)100bs marker, (2.)Uncut pSB1A3, (2.)Uncut pSB1A3, (3.)Cut pSB1A3; (b.) (1.)100bps marker, (2.)Dephosphorelation of Pst1 cutting site in pSB1A3.
+'''
+　　When we confirm the PSB1A3 that contain OmpC promoter. And we amplify it, used Pst1 to cutting off the sequences and run the gel electrophoresis. Due to the cutting form plasmid will change circular to linear form caused molecular weight increasing so the gel electrophoresis speed will slow down and stop at the upper strata  show at the Fig.3-a.
-When we confirm the PSB1A3 contain OmpC. And we amplify it, used pst1 to cutting off the sequences and run the gel electrophoresis. Due to the cutting form plasmid will change circular to linear form caused molecular weight increasing so the gel electrophoresis speed will slow down and stop at the upper strata show at the Fig.3-a.
+　　In order to improve the successful rate of putting insert into the vector we treated, Pst1 digested pSB1A3 with calf  Intestinal Alkaline Phosphatase (CIP) to remove the phosphate group thus reducing the chance of self ligation