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

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Experiment 2 (Jan 20,09)

Part 1: Electrophoresis of PCR products

  • 1.5% agarose gel used.
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


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.


Image:Lab_2_Part_1_agarose_gel.jpg

  • 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.

Image:Lab_2_part_2_agarose_gel.jpg

  • 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. Image:Lab_1_Bacteriophage_Growth_Curve.jpg

  • Figure 1.One-step growth curve for T4 bacteriophage co-incubated with E. coli at 35oC in a shaking water bath

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

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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