Team:AUC TURKEY/Notebook/Protocols

 

§  In a steril environment, the tare of the container should be measured and subtracted from the overall weight.

§  7 grams of LB Agar is put in the container.

§  200 ml distilled water or is put into a graduated cylinder.

§  These two are mixed in a beaker.

§  The opening of the beaker is covered with aliminium folio in such a way that it does not conctact with air.

§  Autoclave tape is sticked on to the aliminium.

§  The beaker is placed in to the autoclave machine.

§  Distilled water or demineralized water is put into the autoclave machine. The water level in the autoclave machine has to be a little higher than the liquid level of the beaker.

§  After closing the lid of the machine, the 90 minute autoclave process is given start.

§  Take out the beaker and add antibiotics if required.

Warnings for the Autoclave!

§  Use only demineralised or disttiled water with the device.

§  Do not open the cover until the manometer drops to zero during the operation.

§  Please do not use the autoclave for other purposes than sterilization and agar.

§  Please do not use the autoclave to sterilize explosive, inflammable and oxidizing materials.

§  Please be cautious when you are closing the lid not to trap your hand.

§  Please beware of the steam exhaust when you are opening to autoclave after sterilization.

§  Please wear protective gloves before removibg materials from the chamber. Do not access the chamber unless the vapor exhaust is finalized.

§  In a steril environment, the tare of the container should be measured and subtracted from the overall weight.

§  7 grams of LB Broth is put in the container.

§  200 ml distilled water or is put into a graduated cylinder.

§  These two are mixed in a beaker.

§  The opening of the beaker is covered with aliminium folio in such a way that it does not conctact with air.

§  Autoclave tape is sticked on to the aliminium.

§  The beaker is placed in to the autoclave machine.

§  Distilled water or demineralized water is put into the autoclave machine. The water level in the autoclave machine has to be a little higher than the liquid level of the beaker.

§  After closing the lid of the machine, the 90 minute autoclave process is given start.

§  Take out the beaker and add antibiotics if required.

Warnings for the Autoclave!

§  Use only demineralised or disttiled water with the device.

§  Do not open the cover until the manometer drops to zero during the operation.

§  Please do not use the autoclave for other purposes than sterilization and agar.

§  Please do not use the autoclave to sterilize explosive, inflammable and oxidizing materials.

§  Please be cautious when you are closing the lid not to trap your hand.

§  Please beware of the steam exhaust when you are opening to autoclave after sterilization.

§  Please wear protective gloves before removibg materials from the chamber. Do not access the chamber unless the vapor exhaust is finalized.

§  Transfer 500 ul LB Broth to 1.5 ml microcentrifuge tubes. This should be done close to a source of fire to prevent contamination.

§  Place the microcentrifuge tubes containing LB Broth in a 42 C heat block for incubation.

§  Take 1 ul plasmid and place them in 1.5 ml centrifuge tubes.

§  Add 50 ul competent cells to the plasmid.

§  Centrifuge them at 3000 rpm for 20-30 seconds.

§  Incubate the cells in ice for 45 minutes.

§  After 45 minutes, heat the tubes in the 42 C heat block for a maximum of 90 seconds.

§  The same tubes should be placed in ice and should be incubated for 5 minutes.

§  Afterwards, 450 ul LB should be added to the cells to complete them to 500 ul.

§  The microcentrifuge tubes are then sticked to the shaker horizantally and shaked for 1 hour with 320 rpm at 37 C.

§  150 ul of the mixture(200 ul for digestion) is then placed on the plate to spread.

§  It is then spread on the plate and the plates are incubated at 37 C for 16 hours.

§  The LB Media should be transferred to 1.5 ml centrifuge tubes.

§  These tubes are then centrifuged at 13,000 rpm for 10 minutes at room temperature.

§  After the centrifuge, the supernatent should be disposed without taking any pellets along with it.

§  The pelleted cells should be suspended in 250 ul Resuspension Solution and the tubes should be vortexed so that no cell clumps remain.

§  250 ul Lysis Solution is added. The tubes are inverted 4-6 times but the inversion shouldn't be done really fast as the Lysis Solution must not be shaken. Also it is smart to heat the Lysis Solution in order to ensure that no pericipitate remain.

§  350 ul Neutralization Solution should be added and the tube should be inverted immediately and throughly by inverting 4-6 times.

§  Centrifuge for 5 minutes to pellet cell debris and chromosomal DNA.

§  Transfer the supernatent to a spin column without taking any of the pellets.

§  Centrifuge the spin column for 1 minutes and discard the liquid at the bottom. Place the column at the same tube again.

§  Add 500 ul Wash Solution and centrifuge for 30-60 seconds. Discard the flow-through and place column back in.

§  Repeat the same process again with 500 ul Wash Solution.

§  Centrifuge for an additional 1 minute.

§  Transfer the columns into 1.5 ml microcentrifuge tubes and leave their caps open for 2 minutes so that the ethanol that they contain dissolves in air.

§  Add 50 ul Elution Buffer to the center of the silica membrane to elute the plasmid DNA. The pipette tip shouldn't the membrane. Incubate for 2 minutes and centrifuge for 2 minutes afterwards.

§  Discard the spin column and store at -20 C.

§  Take the average of the nucleic acid concentrations measured by the spectrometer.

§  Divide 500 by the DNA average.

§  Add 5ul Ne Buffer.

§  Add 0.5ul BSA Buffer.

§  Add 1 ul of the enzymes with barrier tips.

§  If you cut with EcoR1 and SpI, it will be up stream.

§  If you cut with Xbal and Pst1, it will be down stream.

§  Subtract the amount of DNA from 42.5 ul. This result will be the amount of NFW used.

§  Add the NFW with barrier tips and do one pippetting while taking the NFW.

§  Then the DNA is put into the PCR and is left there for 30 minutes.

§  2ul up stream is put into a eppendorf.

§  2ul down stream is also added.

§  2ul plasmid is mixed in as well.

§  2ul Taq Buffer is inserted to the mixture.

§  1ul T4 DNA ligase is then added with barrier tips.

§  11ul NFW is added with barrier tips and should be pipetted once.

§  Then the DNA is put into the PCR and is left there for 30 minutes.

§  Mix 100ml TAE and 0,8 gram agarose in a glass beaker.

§  The mixture is then heated in a microwave for 3 minutes.

§  Afterwards, 3,6 ul EtBr is added and the beaker is mixed on a magnetic mixer.

§  Mold them and wait for 20 minutes fort he gel to harden.

Procedures for Western Blot

§  Western blots allow investigators to determine the molecular weight of a protein and to measure relative amounts of the protein present in different samples.

§  1) Proteins are separated by gel electrophoresis, usually SDS-PAGE.

§  2) The proteins are transfered to a sheet of special blotting paper called nitrocellulose, though other types of paper, or membranes, can be used. The proteins retain the same pattern of separation they had on the gel.

§  3) The blot is incubated with a generic protein (such as milk proteins) to bind to any remaining sticky places on the nitrocellulose. An antibody is then added to the solution which is able to bind to its specific protein. The antibody has an enzyme (e.g. alkaline phosphatase or horseradish peroxidase) or dye attached to it which cannot be seen at this time.

§  4) The location of the antibody is revealed by incubating it with a colorless substrate that the attached enzyme converts to a colored product that can be seen and photographed.

 

Procedures of Sonication

 

1) Resuspend pellet of 10ml cell culture in 1ml lysis buffer (or 100ml bacterial culture for very low expression level).

 

Suggested Lysis buffer : 140mM NaCl; 2.7mM KCL; 10mM Na2HPO4; 1.8mM KH2PO4; pH 7.3 (PBS) 

                                    or 100mM NaCl; 25mM TrisHCl; pH 8.0 

                                    optional 0.02% NaN3 (azide) 

                                    optional protease inhibitors

 

Optional additives to the lysis buffer 

a) 1mM PMSF or protease inhibitor cocktail 1:200 (cocktail for bacterial cells #P-8849 from Sigma) 

b) Dnase 100U/ml or 25-50ug/ml (SIGMA DN-25). Incubate 10min 4°C in the presence of 10mMMgCl2. 

c) Lysozime 0.2mg/ml. Incubate 10min 4°C. 

d) ßME, DTT or DTE  up to 10mM for proteins with many cysteines. 

e) 0.1-2% Triton X-100, NP40; or any other detergent that do not affect the biological activity of your protein. 

f) 10% glycerol (for stabilization of the protein and prevention of aggregation).

 

2) Sonicate in ice bucket 3 x 10sec or more if the cells are not completely disrupted (Lysis is complete when the cloudy cell suspension becomes translucent. Avoid protein denaturation by frothing).

 

3) Spin 5min 13000rpm 4°C. Separate soluble proteins (supernatant) from insoluble or inclusion bodies proteins (pellet). Use supernatant for next step. Keep sample of 40ul of supernatant for PAGE-SDS and Western blot: soluble proteins

 

4) Resuspend pellet in another 1ml lysis buffer and keep sample of 40ul for PAGE-SDS and Western blot: insoluble proteins, or unlysed cells. 

Procedures for Competent Cell Preparation

 A. Preparing glassware and media eliminate detergent

1. Autoclaving glassware filled 3/4 with DD-H2O to remove most detergent residue

2. Media and buffers in detergent free glassware and cultures grown up in detergent free glassware

B. Preparation of the competent cells

Reagents:

-Glycerol stock

-LB plate

-MgCl2-CaCl2 solution

-MgCl2-CaCl2 solutuion

-MgCl2‧6H2O 3.25g

-CaCl2‧2H2O 0.6g

Add H2O to 200ml

-100mM CaCl2

-100mM CaCl2 solution

-CaCl2‧2H2O 2.95g

Add H2O to 200m;

-80% glycerol

-Liquid nitrogen

DAY1:

1. Flame the metal inoculating loop until it is red got and then cools it down

2. Scrape off a portion from the top of the frozen glycerol stock [DO NOT THAW]

3. Streak it onto the LB plate

4. Put the stock back to -80C immediately

5. Leave the plates for 5 minutes and place them upside down in the 37C incubator for 16-20 hours

 DAY 2

6. Pick a single colony into 5ml of LB medium

7. Inoculate the culture overnight at 37C with shaking at 250rpm

 DAY3

8. Inoculate 100ml LB medium with 1ml of saturated overnight culture

9. Shake at 37C until OD600=0.4 (usually 2-3 hours)

10. Place in an ice bath for 10 minutes [ After this point the cells should never

touch anything that is warm]

11. Pre-cool solution, centrifuge, pipette tips, falcon, eppendorf

12. Transfer the culture into two pre-chilled 50ml falcon

13. Centrifuge at 2700x g for 10 minutes at 4C

14. Remove the medium, resuspend the cell pellet with 1.6ml ice0cold

100mM CaCl2 by swirling on ice gently

15. Incubate on ice gor 30 minutes

16. Centrifuge at 2700x g for 10 minutes at 4C

17. Remove the medium, resuspend the cell pellet with 1.6ml ice-cold 100mM CaCl3 by swiriling on ice gently

18. Incubate on ice for 20 minutes

19. Combine cells to one tube and add 0.5 ml ice-cold 80% glycerol and swirl to mix

20. Freeze 100ul aliquots in liquid nitrogen

21. Store in -80C