Team:UB Indonesia/Protocols

BRAWIJAYA UNIVERSITY

PROTOCOL De(Ngue)tective UB_Indonesia iGEM 2015

1. Preparation

a. Medium

Luria Bertani Agar

- Add 40g Luria Bertani agar to 1L demineralized water

- Sterilize in 121°F 0.1 Mpa for 15 minute with autoclave

Luria Bertani Broth

- Add 25g Luria Bertani agar to 1L demineralized water

- Sterilize in 121°F 0.1 Mpa for 15 minute with autoclave

- Chemically reagent

b.1. Competent E. coli cells CCMB pH 6.4

- Prepare a chemicaly substances:

80mM CaCl2 2H2O (11.8g/L)

20mM MnCl2 4H2O (4.0g/L)

10mM MgCl2 6H2O (2.0g/L)

- Dissolve all in 10% glycerol (100mL/L)

- Sterilize in 121°F 0.1 Mpa for 15 minute with autoclave

- Incubate on ice for 30 minutes

- Add 10mM Potassium Acetate pH 7 (10mL of a 1M stock/L)

- Adjust pH to 6.4 used 0.1N HCl

- Store at 4°C untill used

b.2. SOC media for 1L

- Prepare a SOC component:

5g Yeast Extract

20g Tryptone

0.584g NaCl

0.186g KCl

2.4g MgSO4

- Adjust pH tp 7.5 (25mL 1M NaOH/L)

- Sterilize in 121°F 0.1 Mpa for 15 minute with autoclave

- After cooling medium to less than 50°C, add 20mL filter sterilized 20% glucose solution

b.4. TE buffer

b.3. Ligation adjustment buffer

2. E. coli BL 21 subculture

- Prepare a petri dish, tube, LB agar, ose, bunsen, lighter, alcohol 70%, and E. coli BL 21

- Sterilize the area for minimize the contamination and do this step in around of bunsen

- Pour the liquid LB agar into petri dish and make it solidly

- Streak the E. coli BL 21 use ose in around the medium

- Incubate in 37°C for 24h

3. Make a competent E. coli cells

- Pick frozen E. coli BL 21 cell, dissolve in 10-20µL LB, plate on an LB Plate without antibiotics

- Pick a single colony from the plate and grow overnight in 10mL of LB without antibiotics

- Dilute 1mL overnight culture into 100mL LB broth

- Prepare the fresh solutions

Filter Sterilize All Solutions (Make sure bottles are sterile)

0.1M MgCl2 = 10.16g MgCl2 in 500mL

FW: 203.31 g

0.05M CaCl2 = 3.67g CaCl2 in 500ml

FW: 147.02g

0.1M CaCl2 = 2.94g CaCl2 + 28mL Glycerol + 172mL ddH20

- Keep solution on ice

- Grow the cells to Optical Density (OD) 600 = 0.3 – 0.4 (should take approx. 3 to 4 hrs after innoculate with 2.5 ml of cells. The OD frequently of sample should be checked, taking out 1mL)

- Split the preparation into 2 large sterilized centrifuge bottles

- Centrifuge at 4000 rpm for 10 minutes in 4°C

- Pour off supernatant and redissolve each pellet in 50mL 0.1M MgCl2 on ice

- Incubate the cells on ice for 7-10 minutes

- Centrifuge at 4000 rpm for 10 minutes in 4°C

- Dissolve each pellet in 50 ml of 0.5M CaCl2 on cold room

- Incubate on Ice for 20 minutes in cold room

- Centrifuge at 4000 rpm for 10 minutes in 4°C

- Dissolve in 20mL of the 0.1M CaCl2 with glycerol on cold room

- Once dissolved, dispense 500µL aliquots that have been pre-chilled on ice into 1.5mL eppendorf tubes

- Put on dry ice for 1.5 minutes before throwing them in the -70°C freezer

- Store at -70°C Freezer

- Test the cells by performing a mock transformation with a plasmid of known concentration (Use specific dilutions of the vector such as, 1ng, 10ng, 100ng, 500ng,1µg) to determine the transformation efficiency

- Note: rasio of 0.1M MgCl2, 0.5M CaCl2, and 0.1M CaCl2 with gliserol should be 50:50:20

4. Transformation

- Start thawing the competent Escherichia coli cells on ice

- Add 50µL of thawed competent Escherichia coli cells into pre-chilled 1.5mL tube, and also take a same for the control

- Add 2µL of resuspended DNA to the 1.5mL tube.

- Pipet up and down a few times, genlty

- Make sure to keep the competent Escherichia coli cells on ice

- Close tube and incubate the competent Escherichia coli cells on ice for 30 minutes

- Heat shock the competent Escherichia coli cells by immersion in a pre-heated water bath at 42°C for 90 seconds

- Incubate the competent Escherichia coli cells on ice for 5 minutes

- Add 200µL of SOC media (make sure that the broth does not contain antibiotics and is not contaminated) to each transformation

- Incubate the competent Escherichia coli cells at 37°C for 2h while the tubes are rotating or shaking (note: this incubation time helps in transformation efficiency, especially for plasmid backbone with antibiotic resistance other than ampicillin)

- Label two petri dish with LB agar and specific antibiotic(s) with the part number, plasmid backbone, and antibiotic resistance. Plate 20µL of the transformation onto the dishes, and spread it (for make sure that the competent Escherichia coli cells will be able a single colony)

- For the control, label two petri dish with LB agar (AMP) and plate 20µL of the transformation onto the dishes, and spread it

- Incubate the plates at 37°C for 12-14h , making sure the agar silde of the plate is up (if incubate for too long the antibiotics start to break down and un-transformated cells will begin to grow because the resistance enzyme is excreted by the bacteria, and inactivates the antibiotic outside of the bacteria

- Pick a single colony, make a glycerol stock, grow up a cell culture and do a miniprep

- Count the colony on the 20µL control plate and calculate your competent Escherichia coli cell efficiency

5. Miniprep protocol (All protocol steps should be carried out at room temperature)

Here at iGEM we use Qiagen Spin Miniprep Kits for doing small batches of minipreps. If you have never done this protocol before, read the the background information in the handbook; It contains useful information. The following has been reproduced from the handbook and annotated based on experience with the kit. This protocol is designed for purification of up to 20 μg of high-copy plasmid DNA from 1–5 ml overnight cultures of E. coli in LB (Luria-Bertani) medium. For purification of low-copy plasmids and cosmids, large plasmids (>10 kb), and DNA prepared using other methods, refer to the recommendations on page 37.

- Prepare the materials below:

- Qiagen Miniprep Kit

- Cell Culture

- 1.6mL Microcentrifuge tubes (2 per a miniprep)

- TE (1:10)

- Spin the cell culture in a centrifuge to pellet the cells, empty the supernatant (media) into a waste collection container

- Resuspend pelleted bacterial cells in 250 µl Buffer P1 (kept at 4 °C) and transfer to a microcentrifuge tube. No cell clumps should be visible after resuspension of the pellet. Important: Ensure that RNase A has been added to Buffer P1.

- Add 250 μl Buffer P2 and gently invert the tube 4–6 times to mix. Do not vortex, as this will result in shearing of genomic DNA. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 min.

- Add 350 μl Buffer N3 and invert the tube immediately and gently 4–6 times. To avoid localized precipitation, mix the solution gently but thoroughly, immediately after addition of Buffer N3. The solution should become cloudy.

- Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge. A white pellet will form.

- Apply the supernatants from step 4 to the QIAprep spin column by decanting or pipetting.

- Centrifuge for 30–60 s. Discard the flow-through. (Optional): Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30–60 s. Discard the flow-through.

This step is necessary to remove trace nuclease activity when using endA+ strains such as the JM series, HB101 and its derivatives, or any wild-type strain, which have high levels of nuclease activity or high carbohydrate content. Host strains such as XL-1 Blue and DH5α™ do not require this additional wash step.

- (Although they call this step optional, it does not really hurt your yield and you may think you are working with an endA- strain when in reality you are not. Again for this step, spinning for 60 seconds produces good results)

Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30–60 s.

Spinning for 60 seconds produces good results.

- Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer.

IMPORTANT: Residual wash buffer will not be completely removed unless the flow-through is discarded before this additional centrifugation. Residual ethanol from Buffer PE may inhibit subsequent enzymatic reactions. They are right about this.

- Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer EB (10 mM Tris•Cl, pH 8.5) or water to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.

- Notes:

Heating the elution buffer to 55°C prior to loading on the column can slightly increase yields.

Similarly, doing the elution in two steps (first a 30 μL elution and then a 20 μL dilution) can also slightly increase yields.

6. Restriction Digest

When using parts for 3A assembly, or testing the quality of a part you'll need to run a restriction digest. We recommend using the following protocols. estimated time: 30 min. active, 50 min. incubation

- Prepare this materials below:

• Ice and bucket/container

• (1) 8-tube strip, or (3) 0.6ml thin-walled tubes

• BioBrick Part in BioBrick plasmid (Purified DNA, > 16ng/uL)

• dH2O

• NEB Buffer 2

• BSA

• Restriction Enzymes: EcoRI, SpeI, XbaI, PstI

• Thermal cycler

- Keep all materials on ice

- For single reaction, add 250ng of DNA to be digested, and adjust with dH20 for a total volume of 16uL

- Add 2.5ul of NEBuffer 2

- Add 0.5ul of BSA

- Add 0.5ul of EcoRI

- Add 0.5ul of PstI

- There should be a total volume of 20ul. Mix well and spin down briefly.

- Incubate the restriction digest at 37C for 30min, and then 80C for 20min to heat kill the enzymes. We incubate in a thermal cycler with a heated lid

- Run a portion of the digest on a gel (8ul, 100ng), to check that both plasmid backbone and part length are accurate.

7. Ligation

After growing up a part from a distribution or request, you'll want to miniprep it. At iGEM HQ, we use the following protocol.

After following our restriction digest protocol (which uses 250ng of DNA) you may follow these steps for ligation.

- Add 2ul of digested plasmid backbone (25 ng)

- Add equimolar amount of EcoRI-HF SpeI digested fragment (< 3 ul)

- Add equimolar amount of XbaI PstI digested fragment (< 3 ul)

- Add 1 ul T4 DNA ligase buffer. Note: Do not use quick ligase

- Add 0.5 ul T4 DNA ligase

- Add water to 10 ul

- Ligate 16C/30 min, heat kill 80C/20 min

- Transform with 1-2 ul of product

- For linearized plasmid backbones provided by iGEM HQ, a plasmid backbone with an insert of BBa_J04450 was used as template. As a result any red colonies that appear during your ligation may be due to the template as a background. Digesting with Dpn1 before use should reduce this occurrence.

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