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From 2010.igem.org

Contents 1 10/19/10 2 10/19/10 3 IGEM Protocols/Restriction Digest 3.1 Materials 3.2 Protocol 3.3 New restriction and Ligation 4 Using electrocom cells/DNA for electroporateion 4.1 Tubes labeled A-D

10/19/10

Today we are doing large scale DNA extractions from E-Coli Recovering t9002 and lac-z

first taking the OD of each tube

after Blacking spectrometer I will run a sample of each tube.

E-coli w/T9002 from 10/15/10 has an OD of .355 @600um

E-Coli w/LacZ from 10/15/10 has an OD of 3.49 @ 600um

Streaked E-coli w/T9002 from 10/12/10 has an OD of .363 @600um

1. 2 Streaked E-Coli w/LacZ from 10/15/10 has an OD of 3.26 @ 600um

after I took 4 bottles from spec to hood and removed 5ml from each tube placing into 50 cc tubes with LB/Amp and then placed them int 37C inculbator

Then placed the 4 tubes extracted from and placed them into centrefuge to spin down to extract DNA.

Then following protocol from pg 19. After extracting the DNA I placed into fridge in back Room @ 4C.

Then made LB Broth in 3 1Lt flasks weighing 10 g in each LB mix then adding 500 ml to it, placed into microwave one time on beverage setting then into autoclave on liquid setting 121 C @ sw

jhull

10/19/10

I followed the protocol below

I changed my pipette tip each time to prevent cross contamination

IGEM Protocols/Restriction Digest

From partsregistry.org At iGEM HQ we use this protocol for restriction digests along with enzymes purchased from NEB.

Materials

• PCR tube

• dH20

• Enzymes (EcoRI, XbaI, SpeI, PstI)

• BSA

• Enzyme Buffer (NEBuffer 2)*

Notes: You should keep all materials on ice.

Protocol

1. Add 500n~ of DNA to be digested, and adjust with dH20 for a total volume of 42.5ul.

2. Add 5ul ofNEBuffer 2 to the tube.

3. Add 0.5ul of BSA to the robe.

4. Add l ul of your first enzyme

5. Add l ul ofyour second e~e.(~¢,

6. There should be a total volume of 5(iul. Mix well and spin down.

7. Incubate the restriction digest at_3_7C for 30miri, and then 80C for 20min to heat kill the enzymes. l~e incubate in a thermocycler with a heat~-d lid

8. Run a portion of the digest on a gel, to check that both plasmid and part length are accurate. You may also use 2u of the digest (20mg of DNA) for Ligations.

New restriction and Ligation

Joe and I perfomed new restriction and ligation

We did the sam protocol for the first restriction and ligation

The DNA was put into thermocycler 30 min @ 37C and 20 min @ 80C

Stored @ 4.0C in thermocycler

• unsure of the date
• will be used for electrophoresis

Using electrocom cells/DNA for electroporateion

Procedure completed as follows

1. Place 1-5 btl of DNA in a sterile microcentrifuge tube and c[qill on ice. For the pUCl9 control DNA, use l ul (10pg.) (l ul used)

Caution: The DNA must be resuspended in water (rather than TE buffer) to keep the ionic strength to a minimum. Otherwise, arcing might occur during the pulse, which can damage the machine and will result in no transformation and cell death. DNA in ligation or restriction buffer must be precipitated or desalted before electroporation.

2. Gently thaw cells on ice. Use these cells immediately, do not leave them on ice for an extended period of time. Unused cells can be re-frozen for later use, but they will suffer a significant loss of efficiency.

3. Transfer the desired amount of cells to the pre-chilled microcentrifuge tube containing the DNA. It is recommended that 20-40 ~tl (40-80 ~1 if using 0.2 cm cuvettes) be used for a small-scale electroporation (< 100 ng of DNA), and 160 ul for a large-scale or library electroporation. The volume of DNA added should not exceed 5% of the total cell/DNA mixture. Mix the cells gently. Do not mix by pipetting up and down. Leave the cells on ice for 1 minute.

4. After 1 minute, transfer the cells to a chilled cuvette and gently shake them to the bottom of the cuvette. Check the cuvette by looking at the contents from both sides to make sure the cells make contact all the way across the bottom of the chamber without any air bubbles. Do this as quickly as possible, being careful not to warm up the cuvette and cells. Remove the condensation from the outside surfaces of the cuvette with a tissue.

5. Electroporate your sample using the settings listed on the previous page or the manufacturer’s recommended settings.

6. Immediately add 480 ~tl (for small-scale) or 960 btl (for large-scale) of SOC medium to the cells. 7. Transfer the suspension to a 15 ml tube. Incubate it at 37°C in a rotary shaking incubator at 225 rpm for 1 hour to allow expression of the antibiotic resistance.

8. Plate the cells on prewarmed selective plates and incubate overnight at 37°C. We recommend plating 2 different volumes to ensure that at least 1 plate has wellspaced colonies.

Note: For the pUC19 control DNA, make a 1:10 dilution in SOC medium and plate --’- _ 25/ul and 100 ul of cells on LB agar with 100 ug/ml of ampicillin.

Tubes labeled A-D

I’m beginning w/Tube A (DNA incubated)

180 kv - 4.5 ms for tube A

Tube B

same protocol as A

180 kv – 3.4ms for tube B

used 2 ul of DNA instead of 1

put into 37C for incubation

Tub C same protocol as A

180 kv – 3.5ms for tube C

used 2 ul of DNA instead of 1

put into 37C for incubation

Tube D same protocol as A

180 kv – 4.3 ms for tubeD

used 2 ul of DNA instead of 1

put into 37C for incubation

Plating DNA/Cells This will be done for tube A-D

Using Sterile pre-poured plates

add 100ul of DNA/Elec.Com/SOB media

Spread then incubate

dgarvey