Team:METU Turkey/Results Discussion/Cloning

 

CLONING PROTOCOLS

The major point of this project was to assemble the sensor parts to construct CO cell sensor module. For succesful assembly, we had tried many alternative protocols, at the end we construct the desired sensor module with the most efficient methods

Competent Cell

- Inoculate streak plates from liquid stock competent cells Top10(plate with strep) and BL21 DE3(antibiotic free plate) ; incubate overnight at 37 C
- Put 10 mL LB + strep(100ug/ml) + colony into 3 eppendorf. Incubate overnight at 37 C
- Inoculate 200 ul to 1000 ul from overnight culture into 100-500 ml Psi broth. Incubate at 37 C with aeration to A600=0.6-0.7
- Ice 15 min. From this step onward the cells must remain COLD. (4C or on ice), pellet cells in appropriate centrifuge tube 3-5000 x g 5 min
- Discard supernatant and add TfbI 0.4 volume of initial volume, resuspend and ice 15 min.
- Pellet cells in appropriate centrifuge tube 3-5000 x g 5 min
- Discard supernatant and resuspend in 0.04 volume TfbII, ice 15 min and either use immediately or quick freeze at -70C for storage.

Transformation

- Mix 1 uL vector DNA (synthesized genes) and 80 uL competent cells.(1-10 ng vector)
- Incubate in ice bath for 30 min.
- Heat shock for 20 sec. in 42C at heatblock.(not exceed 30 sec)
- Ice bath for 30 sec.
- Add 600ul of SOC medium on bacteria culture.
- Incubate in 200 rpm shaker for 1h
- Inoculate into plates with appropriate antibiotics.

Glycerol Stock Preparation

- Pick at least 5 single each big colonies
- Inoculate each colony into 10 mL subculture incubate at 37 C @ 240 rpm not longer than 12-14 h
- Centrifuge cultures in 5 ml aliquots, discard 4ml of supernatant, resuspend pellet into 1 mL
- Add 176 uL glycerol into each epp to 15% final concentration
- Vortex, aliquot 100 uL samples to cryotube
- Quick-freeze in liquid nitrogen
- Place in -80 C deep freeze

Plasmid purification

Procedure is followed with respect to the instructions of The GeneJET™ Plasmid Miniprep Kit
- Pick a single colony from transformants
- Inoculate in 5 mL LB medium for high-copy or 10 mL for low-copy of LB medium supplemented with the appropriate selection antibiotic.
- Incubate @ 225 rpm not longer than 12-14 h at 37 C .
- Spin @ 4000 rpm for 5 min at 25 C, discard the supernatant and keep pellet.
- Resuspended the pellet with 250 ul Resuspension solution.
- Transfer the cell suspension to eppendorf, add 250 uL Lysis Solution
- Mix throughly by inverting the tube 4-6 times until the solution becomes slightly clear.
- Add 350 uL Neutralization solution
- Mix immediately and throughly by inverting the tube 4-6 times.
- Spin @ 13000 rpm for 5 min to precipitate cell debris and chromasal DNA.
- Transfer the supernatant to spin column 600 uL
- Spin @ 13000 rpm for 1 min, discard the flow-through.
- In same collection tube add 500 uL Wash solution to spin column.
- Spin @ 13000 for 1 mini discard the flow-through.
- Repeat this step with using 500 uL Wash solution then discard the flow-through
- Spin @ 13000 for an additional 1 min to remove residual Wash solution.
- Transfer spin column into a fresh 1.5 mL epp.
- Add 50 uL Elution buffer into center of the spin column membrane to elute the plasmid DNA
- Incubate for 2 min at 25 C spin @ 13000 for 2 min.
- Discard the column and store the purified plasmid DNA at -20 C.
Before digesting the plasmids, the purity and concentartions of plasmids are required. We analyzed these values by Alpha UV spectrophotometer. According to the values in some cases as for small promoter fragments, the isolated plasmids should be concentrated. We processed two methods to precipitate small DNA fragments. One of them was to vacuum the plasmids after isolation in elution buffer. Other method was ethanol precipitation.

Ethanol Precipitation

- Measure out 2X volumes absolute ethanol into DNA sample.
- Incubate at -80°C for 1 hr.
- Centrifuge at a speed of at least 10000 Xg for 30 mins at 0°C, gently aspirate out the supernatant and discard it. (All steps should be done in ice)
- Measure out 750 - 1000 µl of 95% ethanol into the eppendorf tube that is used at first step.
- Centrifuge at a speed of at least 10000 Xg for 10 mins at 4°C, gently aspirate out the supernatant and discard it.
- Dry the pellet in air until white pellet appears.
- Add appropriate volume of water to pellet.
- Resuspend pellet by vortexing/by shaking vigorously.

Restriction Digestion

Prepare total mix in following order with respect to purity values.
- Add water nuclease free to 0.2 mL PCR tube
- Add appropriate 10x buffer (depending on enzyme type fast digest or conventional enzymes)
- Add DNA solution to the tube (ugr)
- Add required unit restriction enzyme for DNA ugr to the tube
- Spin the tube for 5 sec
- Incubate the tube for h at 37 C at heatblock
- For enzyme inactivation incubate tube additional 80 C for 5 min (for fast digest enzymes)
- Purify cut plasmid using instructions of Fermentas PCR purification kit

Validation- Agarose Gel Electrophoresis

-Mix 5 uL DNA + 1 uL loading dye on the parafilm (5:1)
- Load sample to the wells of 1% gel with EtBr
- Adjust the voltage of power supply to 75 V (changes)
- Adjust the time of power supply to 65 min (changes)
- Check transilluminator
- After running of the samples record the gel image

Gel Extraction

The GeneJET™ Gel Extraction Kit instructions are proceeded while extracting the digested DNA fragments from agarose gel.
Before ligating we had tried some more protocols to enhance the efficiency of ligation For efficient ligation of digested fragments, there should not be the phosphates at the end of sites. Then for efficient dephosphorylation of sites of plasmid we treated the fragments with alkaline phosphatase method.This protocol is suitable for removal of 3’ and 5’ -phosphate groups from DNA and RNA.

Alkaline phosphatase treatment

- Prepare the following reaction mixture:
Linear DNA (~3 kb plasmid) 1 µg (~1 pmol termini)
10X reaction buffer for AP used in reaction 2 µl
FastAP™ Thermosensitive Alkaline Phosphatase 1 µl (1 u)
Water, nuclease-free (#R0581) to 20 µl
Total volume 20 µl
- Mix thoroughly, spin briefly and incubate 10 min at 37°C.
- Stop reaction by heating for 5 min at 75°C.

Ligation

Prepare ligation mix in following order and in 1:1 ratio (vector:insert)
- Add nuclease free water
- Add rapid DNA ligation buffer
- Add the calculated amounts of insert elute (1 or more) (elution from gel extraction procedure must be done with nuclease free water)
- Then add from backbone (calculated amount in 1:1 ratio)
- Add T4 DNA ligase ), end up T4 DNA ligase in 10 to 15 uL
- Keep the ligation mixture in room temperature for 5 min.

Transformation

- Mix 1 uL vector DNA (ligated parts) and 80 uL competent cells.(1-10 ng vector)
- Incubate in ice bath for 30 min.
- Heat shock for 20 sec. in 42C at heatblock.(not exceed 30 sec)
- Ice bath for 30 sec.
- Add 600ul of SOC medium on bacteria culture.
- Incubate in 200 rpm shaker for 1h
- Inoculate into plates with appropriate antibiotics.