GeNei TM GFP Cloning Teaching Kit Manual

Transcription

1 Teaching Kit Manual Cat No Revision No.:

2 CONTENTS Page No. Objective 3 Principle 3 Kit Description 7 Materials Provided 9 Procedure 11 Observation & Interpretation 15 Appendix 16 Ordering Information 17 1

3 Objective: To understand the basic concept of cloning and perform some of the steps involved, using the GFP gene (Green Fluorescent Protein). Principle: Molecular cloning or gene cloning involves insertion of a DNA fragment (gene of interest) into a cloning vector. The recombinant vector is subsequently transformed into a suitable host to generate the desired clones. Gene cloning basically involves the following steps: Isolation of vector & insert DNA. Cutting of DNA. Joining of DNA. Amplifying the recombinant DNA. Screening for clones. Isolation of vector and insert DNA: Insert DNA: Foreign DNA is isolated from cells by disrupting lipid membrane with detergents and is subsequently purified by RNase treatment and phenol/chloroform extractions. Insert DNA (having the gene of interest) is then obtained from the purified foreign DNA by shearing, PCR amplification or restriction enzyme digestion. 2 3

4 Vector DNA: Cloning vectors are circular DNA molecules in which DNA fragments/insert are maintained and amplified. Commonly used cloning vector include: Plasmids, Cosmids, Phagemids and Yeast artificial chromosomes etc. Vectors have the following features: o An origin of replication recognized by the host cell replication machinery. o A selection marker that enables the identification of transformants and/or transfectants. o One or multiple restriction endonuclease recognition sites that allow the insertion of gene of interest. Vector DNA purification protocols vary depending upon the type of vector chosen for cloning experiments. Smaller sized cloning vectors like plasmids, cosmids and phagemids are easier to purify and are usually done by alkaline lysis method and subsequent phenol/chloroform extractions. Cutting of DNA: In order to carry out recombination between the vector and the insert DNA, it is necessary to specifically and reproducibly cleave DNA. Restriction enzymes are the scissors of molecular genetics, which recognize specific nucleotide sequences and cut the DNA at these points to generate sticky or blunt ends. Generally, the same restriction enzyme is used to cleave both the insert and vector DNA. Following restriction enzyme digestion of vector, the DNA is dephosphorylated i.e., 5 phosphate group is removed by treating with alkaline phosphatase. This is done to reduce self ligation of vector molecules by T4 DNA ligase. However, this does not prevent ligation of insert to the vector as 5 phosphate groups of insert are intact and thus efficiency of insert ligation to the vector increases. Another method employed to prevent self ligation of vector is to cut the DNA with two different restriction enzymes resulting in mismatched ends that cannot ligate. Joining of DNA: Once the insert and vector DNA have been isolated and cut with specific restriction enzyme(s), they must be ligated. DNA ligase is the glue of molecular genetics that holds the DNA together by creating a phosphodiester bond between the two DNA ends to create a recombinant DNA molecule. When a single restriction enzyme is used to cut the vector and insert DNA, ligation occurs in either direction/orientation. However, insert can be forced to ligate to the vector in the desired orientation, by using two different restriction enzymes to cut both the vector and insert DNA. Amplifying the recombinant DNA: The recombinant DNA thus created is introduced into a compatible host such that the vector DNA is replicated resulting in amplification of the insert DNA. When plasmid DNA is introduced into host cell, the process is referred to as transformation and in case of viral DNA the process is transduction. A successful vector DNA transfer is monitored by selecting for the genetic marker present on the vector. For eg., plasmid vectors usually have dominant selectable markers such as antibiotic resistance genes that confer growth on media containing appropriate concentration of an antibiotic. In case of viral vectors, one observes for formation of plaques. 4 5

5 Screening for clones: The final step involved in cloning is the means to detect the right clone among a population of colonies or plaques. This screening mechanism will depend upon the vector chosen as the cloning vehicle. For example, pbr322 (plasmid vector) carries genes for ampicillin resistance and tetracycline resistance. Both these genes have sites for restriction enzyme that allow for cloning and selection of recombinants by insertional inactivation of one of the genes. Hence, cells with vector alone will be resistant to both the antibiotics, but cells with recombinant DNA will be resistant to only one antibiotic and can be selected by replica plating. Kit Description: In this kit, a vector and an insert DNA (GFP gene isolated from a bioluminescent jelly fish Aequorea victoria) are provided along with the host (E. coli strain DH5α). Using the kit students will carry out the following experiments: Ligation: GFP gene will be ligated to the vector using the enzyme T4 DNA ligase at 16 C. Since, the vector and insert DNA have been cleaved with the same single restriction enzyme, ligation of insert to vector will occur in either direction/orientation. Preparation of competent cells: Host cells will be made competent by using a solution having salts like calcium chloride, magnesium chloride, manganese chloride, etc. Transformation: The ligated sample will then be transformed into competent E.coli cells and plated on LB plates containing ampicillin. Cells having recombinant DNA or self ligated vector DNA will grow since the plasmid carrying ampicillin resistance gene confers antibiotic resistance to the host cells. Screening: The plates will then be visualized under UV light to screen for clones. This is because, the unique 3 dimensional conformation of the GFP releases energy in the form of visible green light on exposure to UV light. Hence, only cells having the insert in the right orientation will glow and these are the clones. However, there will be other clones where in the insert is ligated in the reverse orientation resulting in no expression of the protein. These clones will not glow on exposure to UV light. 6 7

6 KT60 : The kit is designed to carry out 5 ligation reactions of GFP gene to the vector and its transformation into competent E. coli (DH5α) cells. Note: Single batch of competent cells will be prepared, sufficient for 5 transformation experiments. Duration of experiment: Experiment is carried out over a span of 3 days, approximate time taken on each day is indicated below: Day 1 : 3 hours (Revival of host and Ligation of insert to vector) Day 2 : 6 hours (Preparation of competent cells and Transformation) Day 3 : 45 minutes (Observation and Interpretation) Materials Provided: The list below provides information about the materials supplied in the kit. The products should be stored as suggested. Use the kit within 6 months of arrival. Quantity Materials Cat # (5 expts.) Store Ampicillin 100 mg 4 C Host 1 vial 4 C Solution A 40 ml 4 C Control DNA 10 µl -20 C T4 DNA Ligase 5.0 µl -20 C 10X Ligase Assay Buffer 10 µl -20 C Insert 20 µl -20 C Vector DNA 10 µl -20 C LB Broth 15 g RT Agar 5 g RT 1.5 ml vials 25 Nos. RT Materials Required: Equipment : Centrifuge (preferably refrigerated), UV transilluminator (312 nm), Spectrophotometer. Glassware : Capped centrifuge tubes, Conical flask, Petri plates, Test tubes. Reagent : Distilled water. Other Requirements: Crushed ice, Cuvette (of 1 cm path length), Micropipette, Tips, Thermometer, Water bath. 8 9

7 Note: Read the entire procedure before starting the experiment. All microbiological procedures should be done under aseptic conditions. Revive the strain as soon as the lyophilized vial is opened. Prepare competent cells within 3 days of reviving the strain. Entire procedure of preparing competent cells should be done under aseptic conditions. Carry out transformation as soon as the competent cells are prepared. Storage of competent cells may result in poor/no transformants. Transformation efficiency of competent cells should be more than 1 x 10 5 µg of DNA. Efficiency lower than this may lead to lower number of transformants. Pre-cool tubes, pipettes, centrifuge tubes and Solution A, prior to preparation of competent cells. Solution A supplied is sterile, handle under aseptic conditions. 5 ligation reactions and transformations are to be done simultaneously. Ensure that all the required components are ready prior to starting the experiment. For preparation of media, antibiotic, etc., refer appendix. Procedure: Day 1: Revival of Host 1. Break open the lyophilized vial, add 0.1 ml of LB broth. 2. Streak a loopful of the suspension onto LB plate (in duplicates). 3. Incubate the plates at 37 C, overnight. Ligation of Vector to Insert 4. Thaw the ligase assay buffer, vector and insert DNA. Note: Thaw the ligase assay buffer vial on ice, store at -20 C immediately after use. 5. Set up ligation reaction as follows: Water : 11 µl Vector DNA : 2 µl Insert DNA : 4 µl Ligase assay buffer : 2 µl T4 DNA ligase : 1 µl Mix the contents by tapping gently and incubate at 16 C waterbath, overnight. Note: Set up five ligation reactions simultaneously. Day 2: Preparation of Competent Cells 6. Pick moderate sized colonies from the LB plate and inoculate into 100 ml of LB broth (in a 1 litre conical flask). 7. Incubate at 37 C, in a shaker. Grow until OD A 600 reaches 0.3, this takes about 2-3 hours. 8. Chill the culture flask on ice for minutes

8 9. Transfer the culture aseptically into sterile centrifuge tubes and spin down at 6000 rpm for 8 minutes, preferably in a refrigerated centrifuge at 4 C or spin at Room temperature (RT). 10. Discard the supernatant. 11. Resuspend the cell pellet very gently in small volume of ice-cold solution A (approximately 2 ml), using a pre-chilled pipette. Care must be taken not to remove the tubes from ice during resuspension. Add remaining 33 ml of solution A, resuspend gently. 12. Keep the centrifuge tubes on ice for 20 minutes. Centrifuge at 3500 rpm for 15 minutes at 4 C or spin at RT. 13. Discard the supernatant and chill the tube on ice. Resuspend the pellet in 3 ml of ice-cold solution A. Note: Resuspension is to be done gently as cells are very fragile at this stage. 14. Aliquot 0.6 ml of the above suspension into 5 different pre-chilled 1.5 ml vials, aseptically. 15. Heat inactivate the ligated samples at 65 C for 10 minutes. Spin at 5000 rpm for 2 minutes and keep the vials on ice. 16. Aliquot 2 µl (10 ng) of control DNA each into 5 prechilled 1.5 ml vials. Transformation: Note: Use single aliquot of competent cells (0.6 ml) for one set of transformation experiment. 17. Take a single vial of ligated sample and control DNA. To this add 200 µl each of competent cells. Tap the vials gently and incubate on ice for 20 minutes. 18. Label the remaining 200 µl of competent cells as nontransformed cells, place on ice till the plating step (step 24). 19. Tap all the vials gently and incubate on ice for 20 minutes. 20. Heat shock the cells by placing the vial(s) in 42 C water bath for 2 minutes, then return the vials to ice and chill for 5 minutes. 21. Add 0.5 ml of LB broth aseptically to the vial(s) and incubate at 37 C (shaker) for an hour. This is to allow bacteria to recover and express the protein. 22. Pipette 200 µl each from the vials transformed with the ligated mix onto LB-Amp plates and spread thoroughly using spreader/pipette. 23. Pipette 200 µl of LB onto a LB -Amp plate and spread 20 ìl of the cells transformed with control DNA. Label this as control plate. 24. Plate 200 µl of non-transformed cells onto another LB-Amp plate to check for contamination. Label this as non-transformed plate. 25. Incubate the plates at 37 C, overnight. 26. Observe the plates under UV-light (312 nm). Note: If observed add 254 nm of less intensity of glow could vary. 27. Count the number of colonies on the plate and calculate Transformation Efficiency. Note: Steps 17 to 27 explains the protocol for one set of transformation experiment. Carry out 5 such transformations simultaneously

9 Transformation Efficiency is expressed as: No. of transformants/µg of DNA. Observation: Record your observation as follows: Calculation: Transformation Efficiency is: No. of colonies x 1000 ng Amount of DNA plated (in ng) = /µg Plate Growth Glow Control plate Ligated plate Non-transformed plate For example: Amount of DNA transformed = 10 ng Volume of culture plated = 200 µl (of 1 ml) Thus, amount of DNA plated = 2 ng If no. of colonies observed on plating 2 ng = 200 Transformation Efficiency = 200 x = 1 x 10 5 /µg Denote : +ve: when growth or glow is observed. -ve: when growth or glow is not observed. Interpretation: On plating cells transformed with ligated sample/control DNA on LB-Amp plates, growth is observed as the vector DNA confers antibiotic resistance to the host E.coli cells. In contrast, no growth is observed on plating competent cells untransformed with vector DNA as the host E. coli is sensitive to ampicillin. On viewing the ligated plate under UV light, one observes both glowing and non-glowing colonies, indicating ligation of insert to the vector DNA has taken place in either orientation. Hence, about half the number of recombinant colonies would be non-glowing. Control plate will show no glowing colonies as they lack the GFP gene. Bangalore Genei, Bangalore Genei,

10 Appendix: Preparation of LB Agar/broth (1 litre): Dissolve 25 g of media in 800 ml of distilled water. Adjust the ph to 7.0 with 5N NaOH (if necessary) and make up the volume to 1000 ml. Sterilize by autoclaving. For LB agar, add 1.5% agar and autoclave. Ampicillin Preparation: Dissolve 100 mg of ampicillin in 1 ml sterile water to get a stock concentration of 100 mg/ml. Store at 4 C for 2 weeks. Use the antibiotic within this period. For Ampicillin LB media: Add ampicillin to LB broth or agar at a final concentration of 100 µg/ml, when the temperature of the media is around C. Ordering Information: Product Size Cat # 1 Pack Teaching Kit (Consumables for 5 experiments) Sales: geneisales@sanmargroup.com Customer Support: geneitechsupport@sanmargroup.com Following aliquots of media are required for GFP cloning experiment: LB Broth 4 x 5 ml; 1 x 100 ml LB Agar 50 ml LB Agar with ampicillin 300 ml Note: Prepare 100 ml of LB broth in a 1 litre conical flask

11 Notes: Bangalore Genei, Bangalore Genei, 2008