Tissue Culture Flashcards
(31 cards)
DMEM constituents (5)
10% fetal bovine serum (helps cells grow), glutamine (GF), 1% sodium pyruvate (carbon), 1% penstrep and phenyl-red (indicator).
Culture protocol
- Wash cells in PBS (wash media, which contains alpha 1-antitrypsin and would stop the trypsin from working).
- trypsin (proteolytic enzyme)
- incubate
- 8ml media
- Split 1:5 - 2ml + 8ml in each.
Miniprep does what
Remove plasmid from bacterial cell in prep for transfections
Method of mini prep used
Alkaline lysis
Alkaline lysis protocol
E.coli colonies grown and picked, grown in kanamycin enriched broth. This inhibits the growth of any other bacteria in the culture as they lack protein needed to grow.
Cells then lysed with alkaline buffer (containing strong base and a detergent) and centrifuged to separate.
Alkaline lysis solution I - Re-suspension
Glucose - maintains osmotic pressure
TRIS acts as buffer
EDTA - chelates Ca and Mg which are divalent cations required by DNAses and destabilises the cell membrane.
Primes for soln 2.
Alkaline lysis solution II - Lysis
NaOH - disrupts H bonds and makes ssDNA.
SDS - solubilises the proteins. This solution lyses the cells.
Alkaline lysis solution III - Neutralisation
Potassium acetate - decreases alkalinity, renatures plasmid DNA.
Glacial acetic acid (keeps pH balance).
dH20 (plasmid DNA dissolves in it).
This is the NEUTRALISATION STEP.
Washing stage - Alkaline Lysis
Isopropanol and ethanol used to wash off salts and clean DNA.
Transfection Protocol
Introduction of foreign DNA into the nucleus of eukaryotic cells. We carry out TRANSIENT TRANSFECTION - genes only expressed for 24-96 hours.
When do we transfect cells and why?
Cells transfected at 40-80% confluencey since actively dividing cells take up DNA better than quiescent cells.
Methods of transfection
Electroporation (+ve charge in membrane allows DNA to cross and become trapped), microinjection (used for stem cells, micromanipulator and microscope used) and lipofection (liposomes fuse to cell membrane and release DNA inside cell. Used in oncology research).
Calcium phosphate transfection (easy and cheap, precipitate taken up into cells. Can’t be used for in vivo transfection.)
DEAE-dextran is a cationic polymer which allows DNA to be taken into cell.
Media used in transfection
Optimem.
Protocol for transfection
Wash cells in PBS
2ml Trypsin, incubate then add 3ml DMEM.
Centrifuge then add 5ml optimem
- Add to electroporation curettes, with GFP DNA and zap.
Add DMEM and put back in a dish for a couple days.
After transfection we
Prepped a cell lysis buffer then lysed the cells.
Cell lysis buffer does what
harvest cell from plate then use buffer to lyse the cells so we can study the proteins.
Lysis buffer contains
Tris HCl (buffer prevents protein denaturation)
Sucrose (osmotic pressure)
EDTA and EGTA (protease inhibitors, prevent oxidative damage)
Sodium fluoride and Sodium Pyrophosphate are also protease inhibitors.
Triton X solubilises the solution and Beta-mercaptoethanol reduces disulphide bridges and loosens the proteins.
Protocol - cell lysates
Make up lysis buffer and add beta mercaptoethanol, then pour this over the cells and scrape them into eppendorf. Centrifuge and retain supernatant.
PCR Protocol
3 steps: Denaturation, Annealing and Extension. Annealing and Extension carried out around 30 times.
Denaturation
DsDNA templates heated up to 95 degrees.
Annealing
Products cooled to 61 degrees, primer 1 anneals to 5’ end and 2 and 3 to 3’ end. 1 and 2 stick to wt DNA and 1 and 3 to mut. DNA.
Extension
heated to 72 degrees - Taq polymerase extends DNA strand by reading strand and recruiting dNTPs.
Visualisation
Sybr safe stain added which binds to the DNA and fluoresces under UV light so results can be visualised. DNA ran on agarose gel - biggest bits of DNA move shortest distance.
Western Blot aim
To determine presence of transfected GFP in COS-7 monkey kidney cells.
