Team:Chiba/jk/β/week 3

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Week 3
>last week

31 August, 2008

 * Transformation
 * Competent Cells : XL10G
 * BBa_K084007(Plac+RBS+LasI)
 * BBa_K084008(Plac+RBS+RhlI)


 * ---> We saved these plates with a refrigerator.


 * Digestion
 * BBa_I9026(2007)
 * BBa_I9030(2006)
 * BBa_S03154(2007)
 * BBa_R0010(2007)




 * --->(1/9)Gel Check


 * (30/8)--->Mini prep


 * --->Digestion test
 * Plac+RBS+RhlI Sample No.2~5
 * BBa_T9002①、②


 * --->Gel Check

1 September, 2008

 * (31/8)--->Gel Check


 * --->Gel extract


 * --->zymo
 * insert-1(I9026) -> 7μL
 * insert-2(I9030) -> 7μL
 * insert-3(S03154) -> 7μL
 * vector-4(R0010) -> 15μL


 * --->SAP
 * vector-4(R0010)


 * --->Zymo
 * vector-4(R0010) -> 20μL


 * --->Gel Check


 * --->Ligation


 * --->Transformation
 * Competent cells : XL10GOLD 30μL
 * Transformed the following and grew on new ampicillin plates.
 * BBa_K084009(Plac+RBS+RhlI+LVA, Amp) -> 628 colonies
 * BBa_K084010(Plac+RBS+CinI+LVA, Amp) -> 500 colonies
 * insert-1(RBS+RhlI+LVA) -> 9 colonies
 * insert-2(RBS+CinI+LVA) -> No colonies on the plate
 * vector-4(Plac, Amp) -> 186 colonies


 * --->(2/9) Colony PCR

Transformation
 * Competent cells : JW1908 40μL
 * Transformed the following and grew on new ampicillin plates.
 * BBa_K084007(Plac+RBS+LasI)
 * BBa_K084008(Plac+RBS+RhlI)
 * BBa_T9002(Ptet+RBS+LuxR+GFP)


 * --->(2/9)Liquid Culture

2 September, 2008

 * (31/8)--->Gel Check


 * (1/9)---> Colony PCR
 * Colony PCR of 8 colonies from ligation plates (1/9:(1)BBa_K084009(R1~R8),(2)BBa_K084010(C1~C8)) and one from control plate(BBa_F2620(2007)).




 * 95℃,5min -> ( 95℃,1min -> 52℃,1min -> 72℃,1min )･･･25cycles -> 72℃,10min -> 6℃

--->Gel Check
 * {|align="justify"


 * [[Image:Chiba-0902-2.JPG]]
 * From left;
 * Plac+RBS+RhlI+LVA
 * R1 -> OK
 * R2 -> Bad
 * R3~R7 -> OK
 * R8 -> Bad
 * R3~R7 -> OK
 * R8 -> Bad


 * }


 * --->(3/9)Mini prep

(1/9)--->Liquid Culture
 * Cultured the following cells (2mL LB-Amp, at 37℃, 7 hours)
 * from transformed plates:
 * BBa_K084007(Plac+RBS+LasI, Competent Cells : JW1908)
 * BBa_K084008(Plac+RBS+RhlI, Competent Cells : JW1908)
 * BBa_T9002(Ptet+RBS+LuxR+GFP, Competent Cells : JW1908)
 * from Glycerol Stock:
 * BBa_S03623(Ptet+RBS+LuxI, Competent Cells : JW1908)

--->(3/9)Phenotype test

Transformation
 * Competent cells : XL10G 30μL
 * BBa_C0161(2007)
 * BBa_C0161(2006)
 * BBa_C0261(2007)
 * BBa_C0261(2006)

--->(4/9)Mini prep

3 September, 2008

 * (2/9)--->Mini prep
 * BBa_K084009(R1, R3~R7)
 * BBa_K084010(C1,C2,C4~C8)
 * BBa_S03154


 * --->Gel Check


 * --->Digestion test
 * BBa_K084009(R1, R3~R7)
 * BBa_K084010(C1,C2,C4~C8)


 * --->Gel Check


 * (2/9)--->Phenotype-test
 * MIX
 * BBa_K084007(Plac+RBS+LasI, Competent Cells : JW1908) -> Sample Name : L1~L4
 * BBa_K084008(Plac+RBS+RhlI, Competent Cells : JW1908) -> Sample Name : R1~R4
 * BBa_S03623(Ptet+RBS+LuxI, Competent Cells : JW1908)
 * BBa_T9002(Ptet+RBS+LuxR+GFP, Competent Cells : JW1908)




 * Incubated for 8hours at 37 degrees
 * Spindown (max rpm, 3 min)
 * The measurement of the intensity GFP(visual judgment)


 * Leaving for 12 at room temperture.
 * Resuspension
 * The measurement of the intensity of GFP by Fluoroskan Ascent 2.5(program:Ascent Software Version 2.6)

4 September, 2008

 * --->Gel Check


 * --->Digestion test


 * --->Gel Check


 * Transformation
 * Competent cells : JW1908
 * Transformed the following and grew on new ampicillin plates.
 * BBa_K084009(R-3,4,6,7)
 * BBa_K084010(C-1,2,6,7)
 * BBa_T9002(2007)


 * --->(5/9)Liquid Culture

5 September, 2008

 * (4/9)--->Liquid Culture
 * Cultured the following cells (2mL LB-Amp, at 37℃)
 * from transformed plates:
 * BBa_K084009(Plac+RBS+RhlI+LVA, Competent Cells : JW1908)(r1, r3~r7)
 * BBa_K084010(Plac+RBS+RhlI, Competent Cells : JW1908)(c1,c2,c4~c8)
 * from Glycerol Stock:
 * BBa_S03623(Ptet+RBS+LuxI, Competent Cells : JW1908)


 * --->Phenotype test
 * MIX


 * Incubated for 8hours at 37 degrees
 * The measurement of the intensity GFP by Fluoroskan Ascent 2.5(program:Ascent Software Version 2.6)


 * Spindown (max rpm, 3 min)
 * The measurement of the intensity GFP(visual judgment) (UV 312nm)

6 September, 2008
Colony-PCR
 * PCR of BBa_I9030 and two control,BBa_F2620(2007)).




 * 95℃,5min -> ( 95℃,1min -> 54℃,1min -> 72℃,1min )･･･25cycles -> 72℃,10min -> 6℃ store


 * --->Gel Check


 * From left;
 * 1   I9030 without LVA  -> OK
 * 2   I9030 Positive Control  -> OK
 * 3   F2620 Positive control  -> OK
 * 3   F2620 Positive control  -> OK

Gel Check Sample DNA : R0010

Digestion Test
 * Sample DNA:

1:R0010,2:S03154,3:J04500,4:C0161,5:C0261