Team:HokkaidoU Japan/Notebook/October2

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Revision as of 17:04, 2 October 2010

October 1

Notebook

October 3

Colony PCR

Fig.1

Performed Colonie PCR for 3 colonies which were incubated over night (Fig.1)
Colony numbers were: 1, 2 and 3
Results showed no insertion
- but when result came, had already done miniprep and prepared Sequencing Master Mix

Miniprep

Used Qiagen kit
Melted in 50 uL TE instead of H₂O

Preparation for Sequencing

Mixed as shown in the table below

5x Sequencing Buffer 1.5uL 24.75 uL Ready Reaction Premix 1 uL 16.5 H2O 5 uL 80 uL total 7.5/sample

Reagent	Amount	Amount for 16.5
5x Sequencing Buffer	1.5uL	24.75 uL
Ready Reaction Premix	1 uL	16.5
H2O	5 uL	80 uL
Total	7.5/sample	121.25

3 Piece Ligation: Retry

Gel Extraction

Gel Extraction of DNAs that had been cut on October 1

Couldn't see T3SS signal's bands, retried digestion like below

Digestion

Reagent	Amount
10x M buffer	10 uL
DW	64
T3SS signal	6
BSA	10
Xba I	9.6
Pst I	0.4
Total	100 uL

-> Electrophoresed with other samples

2 piece ligation of T3SS signal and pSB1C3 for DNA Submission

Fig.2

T3SS signal

Reagent	Amount
10x M buffer	2 uL
DW	12
DNA	3
BSA	2
EcoR I	0.5
Pst I	0.5
Total	20 uL

pSB1C3

Reagent	Amount
10x M buffer	2 uL
DW	13
DNA	2
BSA	2
EcoR I	0.5
Pst I	0.5
Total	20 uL

->Incubated at 37C for 2 hrs

Electrophoresis (Fig.2: λ/HindIII 2uL, T3SS signal, pSB1C3, T3SS signal for 3 piece ligation(applied to 2 wells))

Colony PCR

Fig.3

Did colony PCR of latecomers (No.5 to No.16)(Fig.3)

No.8, 10, 11, 12, 15 looked like good
Transfered No.8, 10, 11, 12, 13, 14, 15, 16(control) to 2 mL LBT (Tetracycline 15 ug/mL) and started incubation

Colony PCR

Fig.4

Fig.5

Did colony PCR to amplify T3SS signal (from BAC clone that has T3SS signal insertion)

Colony solution 7 uL, quick Taq 10 uL, primer(Forward and Reverse) 1.5 uL each
Extention: 90 sec, 40 cycles
Electrophoresis to purify the DNA via gel extraction (Fig.4)
Electrophoresis to estimate the concentration of the DNA (Fig.5: λ/HindIII 2 uL, DNA solution 1 uL)
Concentration was estimated at 40 ng/uL

Digestion

Reagent	Amount
10x M buffer	2 uL
DW	9
DNA	5
BSA	2
EcoR I	1
Pst I	1
Total	20 uL

@@ Line 143: / Line 143: @@
 ==Colony PCR==
-[[Image:HokkaidoU Japan 20101002c.JPG|200px|right|thumb]]
+[[Image:HokkaidoU Japan 20101002c.JPG|200px|right|thumb|Fig.3]]
-Did colony PCR of latecomers (No.5 to No.16)
+Did colony PCR of latecomers (No.5 to No.16)(Fig.3)
 * No.8, 10, 11, 12, 15 looked like good
 * Transfered No.8, 10, 11, 12, 13, 14, 15, 16(control) to 2 mL LBT (Tetracycline 15 ug/mL) and started incubation
+<div style="clear:both;"></div>
 ==Colony PCR==
-[[Image:HokkaidoU Japan 20101002d.JPG|200px|right|thumb]]
+[[Image:HokkaidoU Japan 20101002d.JPG|200px|right|thumb|Fig.4]]
-[[Image:HokkaidoU Japan 20101002e.JPG|200px|right|thumb]]
+[[Image:HokkaidoU Japan 20101002e.JPG|200px|right|thumb|Fig.5]]
 Did colony PCR to amplify T3SS signal (from BAC clone that has T3SS signal insertion)
 * Colony solution 7 uL, quick Taq 10 uL, primer(Forward and Reverse) 1.5 uL each
 * Extention: 90 sec, 40 cycles
-->Did electrophoresis to purify the DNA via gel extraction<br>
+* Electrophoresis to purify the DNA via gel extraction (Fig.4)
-->Did electrophoresis to estimat the concentration of the DNA (Fig.5: λ/''Hin''dIII 2 uL, DNA solution 1 uL)<br>
+* Electrophoresis to estimate the concentration of the DNA (Fig.5: λ/''Hin''dIII 2 uL, DNA solution 1 uL)
-->Concentration was estimated at 40 ng/uL
+* Concentration was estimated at 40 ng/uL
 ===Digestion===
+{|style="text-align:center;" class="protocol"
+!Reagent
+!Amount
+|-
+|10x M buffer
+|2 uL
+|-
+|DW
+|9
+|-
+|DNA
+|5
+|-
+|BSA
+|2
+|-
+|EcoR I
+|1
+|-
+|Pst I
+|1
+|-
+|style="border-top:1px solid #996;"|'''Total'''
+|style="border-top:1px solid #996;"|'''20 uL'''
+|}