Team:Uppsala-SwedenWeek7

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Latest revision as of 05:59, 27 October 2010

Week-7

Preparation for getting the bricks:

(1)Competent cell preparation

(2)Transformation of the Biobricks into DH5-alpha competent cells

(3)Verificiation of the lengths of the biobricks by Gel Electrophoresis after PCR.

Lab Setup

We prepared the Competent cells to transform the Bio-bricks obtained from the registry.

Preparation of competent cells

1.Start with preparing competent cells protocol

2.Pour out the 250mL cultivation solution

250/6 = 41.88 ≈ 42mL

3.Separate into 6 falcon tubes ×42mL

4.3000 g centrifugation at 4℃for 10 mins

5.Liquid taken away from supernatant

6.Then, cells in each tube was resuspended with 1mL CCMB80 buffer

Media was prepared and plated with appropriate Antibiotics.

LB medium

800mL H2O

16g LB-broth powder

 Adjust the medium to pH= 7.49

LB agar medium

800mL H2O

 12g agar

16g LB-broth powder

 Adjust the medium to pH= 7.51

Agar plate preparation with ampicillin

Amipicillin stock concentration=100mg/mL

Volume of agar medium=800mL

Ampicillin concentration required = 0.1mg/mL

Mamp=800mL × 0.1mg /mL= 80mg

Vsoc, with amp= 80mg /100mg/mL= 0.8mL in the 800mL SOC medium

The colonies obtained were amplified by colony PCR. (Refer to the PCR protocol for further details). The PCR product was run on a GEL to verify whether the Bio-bricks are of correct lengths. Having verified the lengths of the Bio-bricks the selected colonies were inoculated over night. The inoculated colonies were extracted.

Details of the biobricks we are using:

Biobricks details

Part BBa_

Name

Length (bp)

Plasmid Length

Total Length

Plasmid

Plasmid number

Plate

Well

Antibiotics

Sequencing

Quality (Gel, Q, P)

Function

F 2621

F2

1158

2079

3237

PSB1A2

All

2

21F

Amp

Confirmed

Bad, OK, OK

Sensor

F 1610

F1

798

3189

3987

pSB1Ak3

AHL Relay

2

24G

Amp & Km

Confirmed

OK, OK, OK

AHL generator

I 746350

F17

237

2079

2316

pSB1A2

1 & 4

2

12C

Amp

Partially

OK, OK, OK

Activator in sensitivity tuner

I 746352

F4

264

2079

2343

pSB1A2

2,3,5 & 6

2

12G

Amp

Partially

OK, OK, OK

Activator in sensitivity tuner

B0015

F5

129

3189

3318

pSB1AK3

All

1

23L

Amp & Km

Confirmed

OK, OK, OK

Terminator

I746360

F6

91

2079

2170

pSB1A2

1 & 4

2

12I

Amp

Confirmed

OK, OK, OK

Lower level promotor

I746364

F7

93

2079

2172

pSB1A2

2 & 5

2

12O

Amp

Confirmed

OK, OK, OK

Lower level promotor

I746365

F8

92

2079

2171

pSB1A2

3 & 6

2

14A

Amp

Inconsistent

OK, OK, OK

Lower level promotor

Q04510

F9

987

4425

5412

pSB2K3

All

1

18B

Km

Confirmed

OK, Low, OK

Inverter

P0412

F10

1308

2079

3387

pSB1A2

All

3

6P

Amp

Confirmed

OK, OK, OK

Lower level repressor

I746361

F11

92

2079

2171

pSB1A2

1,2,4 & 5

2

12K

Amp

Confirmed

OK, OK, OK

Higher level promotor

R0010

F12

200

2079

2279

pSB1A2

4,5 & 6

1

1D

Amp

Confirmed

OK, OK, OK

Regulatory promotor

I0460

F16

969

3189

4158

pSB1AK3

4,5 & 6

2

24I

Amp & Km

Partially

OK, OK, OK

AiiA generator

I13504

F13

875

2079

2954

pSB1A2

1 (GFP)

1

22I

Amp

Confirmed

OK, High, OK

GFP Generator

I13507

F14

861

2079

2940

pSB1A2

2 (RFP)

1

22O

Amp

Confirmed

OK, High, OK

RFP Generator

E0430

F15

878

2079

2957

pSB1A2

3 (YFP)

1

8I

Amp

Confirmed

OK, High, OK

YFP Generator

B0034

F3

12

2079

2091

pSB1A2

4,5 & 6

1

2M

Amp

Confirmed

OK, High, OK

RBS

@@ Line 1: / Line 1: @@
 {{Template:Uppsala}}<!--Do not remove the first and last lines in this page!-->
 == Week-7 ==
+== Preparation for getting the bricks: ==
+(1)Competent cell preparation
+(2)Transformation of the Biobricks into DH5-alpha competent cells
+(3)Verificiation of the lengths of the biobricks by Gel Electrophoresis after PCR.
 '''Lab Setup'''
@@ Line 44: / Line 54: @@
  Adjust the medium to pH= 7.51
-'''SOC-mechice'''
-g trypton
- 5h yeast extract
- 0.5g NaCl
- 0.372g KCl
-.2g Glucose
- 400mL H2O
- Separated half of the mixture in to 2 bottles and added 475mL of H2O in each bottle, which makes the total volume of 950mL.
-Ampicillin stock preparation
-Tube weights 13.28747g
-Tube with ampicillin weights 16.65591g
-Amipicillin weights 3.36844g
-Target concentration 100mg/mL=0.1g/mL
-Dissolved in 33.684mL H2O and mixed well. Filtered with 0.22μm filter and poured 1mL in each tube. Frozen the tubes in -8 degree freezer.
@@ Line 91: / Line 73: @@
 The colonies obtained were amplified by colony PCR. (Refer to the PCR protocol for further details). The PCR product was run on a GEL to verify whether the Bio-bricks are of correct lengths. Having verified the lengths of the Bio-bricks the selected colonies were inoculated over night. The inoculated colonies were extracted.
 == Details of the biobricks we are using: ==