User:Mark X. Ling/Notebook/Micb 323/2009/01/20

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Part 1: Electrophoresis of PCR products
Lane 1      | Lane 2       | Lane 3 10μL KCl    | 0μL KCl      | 25μL KCl Lane 4      | Lane 5       | Lane 6 70μL KCl    | MassRuler    | DNA1+Primer1 Lane 7      | Lane 8       | Lane 9 DNA2+Primer1 | DNA1+Primer2 | DNA2+Primer2 Lane 10     | Lane 11      | Lane 12 No DNA      | MassRuler    | 7.5μL MgCl2 Lane 13     | Lane 14     | Lane 15 0μL MgCl2   |20μL MgCl2   | 60μL MgCl2
 * 1.5% agarose gel used.

of all the KCl adjustment. only the positive control showed result. 2.5X the KCl will inhibit the denaturation of 380bp long sequence and stop the PCR amplification of the 380bp sequence.


 * Figure 1. Photograph of Agarose Gel for Part 1; showing the result of PCR using DNA from S.rimosus and S.griseus Using two otrA primer sets.
 * Figure 2.Photograph of Agarose gel for Part 2; showing the effect of Varying KCl Concentration from PCR of S.rimosus with primer set 1.

Part 2: Ligation of exp.1 PCR product

 * pCR 2.1 vector used; T4 ligase used.
 * note: T4 ligase can ligate blunt ends.

Part 3: Bacteriophage growth curve result
experimental flask was diluted by 10-4 before beginning the co-incubation. plaque counts: T0(cell pellet + unadsorbed): Undiluted: 1)47 2)40 10-1:1)1 2)3 10-2:no mark look like plaque, no plaque T0(supernant/unadsorbed): Undiluted: 1)23 2)29 10-1:1)1 2)2 10-2:1) 2) T0(cell pellet): Undiluted: 1)23 2)29 10-1:1)1 2)2 10-2:1) 2) T=5: Undiluted: 1)61 2)62 10-1:1)11 2)3 10-2:1) 2)1 10-3:1) 2) T=10: Undiluted: 1)67 2)59 10-1:marking did not look like plaques 10-2:1)5 2)6 ( 10-3:1) 2) T=15: Undiluted: 1)53 2)74; 10-1:1)5 2)5 10-2:1)1 2) 10-3:1) 2) T=20: 10-1:1)6 2)10 10-2:1)2 2) 10-3:1) 2) 10-4:1) 2) T=30: 10-1:1)120 2)132 10-2:1)23 2)28 10-3:1) 2) 10-4:1) 2) T=40: 10-1:1)298 2) 10-2:1)34 2)41 10-3:1)4 2)1 10-4:1) 2)1 T=50: 10-1:1) 2) 10-2:1)32 2)44 10-3:1)3 2) 10-4:1) 2) T=60: 10-1:1) 2) 10-2:1)61 2)43 10-3:1) 2) 10-4:1) 2) T=70: 10-1:1) 2) 10-2:1)95 2)81 10-3:1)5 2)9 10-4:1)31 2)27
 * Figure 1.One-step growth curve for T4 bacteriophage co-incubated with E. coli at 35oC in a shaking water bath

Post Lab comments

 * PCR itself went well.
 * the manipulation of KCL, too extreme, only control had any product.
 * part 1 of the PCR showed good band
 * resolution of bands could be better.


 * the growth curve of the bacteriophage look very good.


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