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From 2010.igem.org

Week-8

Preparation for Plasmid construction:

(1) Plasmid Extraction

(2) Digestion and Ligation

(3) Transformation of the ligate

(4) Screening of the Transformant and cheking for the correct length

Plasmid extraction

Fermentas Gene Jet Plasmid Miniprep Kit. Batch Nr. K0503

1.Read the important notes in the kits protocol and make sure all buffers and other solution are ready to use BEFORE STARTING.

Check if there is enough Gene Jet tubes for all your samples.

2.Make sure there is a glycerol stock for each sample is made BEFORE STARTING.

3.Centrifuge 5 min, 5800 rpm, 19 °C

4.Decant the all the supernatant

Purification protocol

NOTE! All purification steps should be carried out in room temperature All centrifugation in 12000 g micro centrifuge

psB1A2 - High copy plasmid psB1AK3 ­- High copy plasmid psB2K3 - Variable copy

Check if the plasmid backbone is high copy or low copy plasmid

For high copy and variable plasmid: Add 5 ml LB-medium For low copy plasmid: Add 10 ml LB-medium

5.Resuspend the pelleted cells with 250 µL Resuspension Solution

6.Transfer the cell suspension to eppendorf tubes

7.Vortex until you have a homogenize solution

8.Add 250 µL Lysis solution. Mix thoroughly by inverting the tubes 4-6 times

9.Add 350 µL Neutralization Solution and mix immediately by inverting 4-6 times

10.Centrifuge for 5 min

11.Transfer the supernatant to the GeneJet Spin column by pipetting .

12.Centrifuge for 1 min. Discard the flow-through and place the column into the same collection tube.

13.Add 500 µL Wash Solution. Centrifuge for 1 min. Discard the flow-through and place the column into the same collection tube.

14.Repeat step 13. After discarding the flow-through, centrifuge for an additional 1 min to remove all the wash solution.

15.Transfer the GeneJET spin column into a new eppendorf tube (which should be properly marked). Add 50 µL Elution Buffer to the center of the GeneJET spin column. Important! Do not make contact the column membrane with the tip. Incubate for 2 min room temp. Centrifuge for 2 min.

16.Pipette up the whole flow through and add it again in the center of the GeneJet Spin Column. . Incubate for 2 min room temp. Centrifuge for 2 min

17.Throw the column. Continue the with DNA concentration, PCR & Gel in spectrophotometer. Store at - 20 °C

Digestion, Purification and Ligation.

Digestion

Calculate the volume from each DNA-extraction you need in order to get a total of 1 µg DNA, based on the plasmid concentration data.

1.Check in the flow chart which Restriction enzymes is required for each, specific BioBrick device. 1 µL RS/ 1µg DNA. Decide a final volume for each digestion sample, which include the:

Autoclaved water Fast Digestion buffer 10x DNA RS1 RS2

2.Add everything in order above, into a eppendorf tube, with the restriction enzymes last.

3.Incubate for 30 min, 37 °C water bath. Note that if the DNA is lower than 500 bp. Namita said it would just be fine continuing =P.

Purification

GeneJET PCR Purification Kit. Fermentas All purification steps should be carried out in room temperature. All centrifugation in 12000 g

1.Add 1:1 volume Binding Buffer (relative for each digestion sample) Mix throughly and check the color. If yellow: OK If Orange or violet: Add 10 µL 3M Sodium pH 5:2

2.Transfer up to 800 µL of the solution to a GeneJET purification column

3.Add 700 µL Wash buffer to the GeneJET purification column. Centrifuge for 1 min

4.Discard the flow-through and place back the purification column back on the collection tube

5.Centrifuge the empty GeneJET Purification column for 1 min. This to completely remove the any residual wash buffer left

6.Transfer the GeneJet Purification column to a new eppendorf tube.

7.Add 50 µL Elution Buffer to the center of the GeneJET purification column. Centrifuge for 1 min

8.Add the flow-through back in the center of the GeneJET purification column and centrifuge for 1 min again.

9.Discard the GeneJET purification column. Next; Ligation step or store at –20 °C

Ligation

3A assembly

1.Add the following components, in a autoclaved eppendorf tube, for 3A assembly in the following order and according to the flow chart.

Biobrick 1 4 µL Biobrick 2 4 µL Vector 2 µL Quick Ligase Buffer 10 µL Quick Ligase 1 µL

2.Incubate in 5-6 min. Done!

3.Next step: Transformation

Protocol for cell transformation, plating

Transformation

1.10 μL from the ligation stock the competent cell tube

2.Incubate 30 min on ice

3.Prepare the 42 °C water bath

4.Heat chock 1 min in 42 °C water bath

5.Add 900 μL LB- medium to the same tube

6.Incubate for 1 h, 37 °C in 250 rpm

7.Centrifuge 5 min, 6000 rpm

8.Remove 800 μL supernatant (In sterile bench from this step)

9.Resuspend the pellet with the remaining solution

Plating

10. Pour the whole resuspended solution on the plates

11.Pour 5 autoclaved glasbeads on the plate.

12.Shake horizontally and shift the position plate relative your hand several times

13.Carefully tip out the beads on beads waste glass.

14.Place in over overnight

PCR/ COLONY PCR protocol

When taking colony from the plate.

Pick 5 different colonies from each plate with 10 µL tip. Put in a eppendorf with 10 µL LB. Take 1 µL of the LB-solution in the PCR tube with the master mix.

Master Mix composition for 1 sample. Multiple each chemical with x samples + 3 in order to get some margin when transferring the mix to the tubes.

1. Add in the following order:

H20 16,22 µL Dream taq Buffer 2 µL MgCl2 0 µL dNTP:s 0,4 µL DMSO 0 µL Primer forward 0,4 µL Primer reverse 0,4 µL Dream Taq 0,08 µL Total 19,5 µL/ PCR sample + DNA 1 µL

2. DANTAQ Program in the PCR machine.

Be aware that we must change certain Program Setup, as elongation time, 1min/Kb, as the biobrick-assembly gets larger and larger.

3. Gel. 1% agarose relative the volume of the sodium boric acid, depending many wells are necessary

4. 1 µL Blue dye on a plastic film + 9 µL of PCR sample well mixed.

5. 200 V were applied to the gel.

Gel image of the biobricks after confirmation of lengths:

   * Outcome:We confirmed the lengths of almost all the transformed cells of the required biobricks to be correct as per above gel image.