Lab Skills

Question 1

Tissue Culture

1. What were the components of DMEM (5)?
2. How often and at what split cells passaged?
3. Why was PBS used to wash off media?
4. What is trypsin?
5. What was the CO2 concentration in the incubator?

Answer 1

1. 10% fetal bovine serum (helps cells grow), glutamine (helps cells grow), 1% sodium pyruvate (carbon source), 1% penstrep (penicillin + streptomycin), phenyl red (already in media - gives cells red colour)
2. Twice a week 1:5 split
3. PBS used to wash cells as inhibitors in media (e.g. alpha-1 antitrypsin) stop trypsin working
4. Trypsin is a proteolytic enzyme (serine protease) which breaks bonds between cells
5. 5%

Question 2

Bacteria Colony Transfer

1. What antibiotic was used in the agar of the bacteria before they were transferred?

Answer 2

1. Bacteria had been plated onto agar with kanamycin present (only bacteria with specific protein could grow)

Question 3

Mini Prep

1. What were the components of Solution I (3)?
2. What were the components of Solution II (2)?
3. What were the components of Solution III (3)?

Answer 3

1. Resuspension of E.coli pellet - Glucose (osmotic buffer), TRIS (buffer), EDTA (needed by DNAses for Ca2+ and Mg2+ and to destabilise cell wall)
2. Used to lyse cells - NaOH (disrupts hydrogen bonds between bases which converts double-stranded DNA into single strand), SDS (detergent which solubilises the proteins)
3. Neutralisation step - Potassium acetate (decreases alkalinity and renatures double-stranded DNA [only plasmid DNA not genomic]), glacial acetic acid (keep pH balanced), dH2O (only plasmid DNA dissolves in it [not genomic DNA])

Protein and chromosomal DNA were removed during the neutralisation step (as insoluble debris was precipitated)
Alcohol precipitation in the end removed salts and small molecules

Question 4

Transfections

1. What is transfection?
2. What split was used to passage cells before transfection and why?
3. What method was used for transfection?
4. What other methods could have been used?
5. What is Optimem?

Answer 4

1. Foreign DNA transfected into nucleus of cells (transcription + translation leads to expression of proteins)
2. Cells passaged 1:3 so they are in active growth phase before transfections
3. Electroporation: creates electric field (positive charge across membrane, and as DNA is -ve charged it flows through pores in the membrane and becomes trapped inside cell)
4. Microinjection: different method to electroporation that is much slower but more effective; Lipofection: use lipofectamine and DNA which combine and allows DNA into cell (efficient but not as cost-effective as electroporation
5. Optimem is a media to keep cells alive that doesn’t interfere with transfection process

Question 5

Prep of Lysis Buffers
Why were the following used in the lysis buffer:
1. TRIS?
2. Sucrose?
3. EDTA + EGTA?
4. Sodium fluoride and pyrophosphate?
5. Triton X-100?
5. Protease inhibitor?
7. b-mercaptoethanol?

Answer 5

1. TRIS: buffer that prevents protein denaturation
2. Sucrose: maintains osmotic pressure so cell doesn’t burst until required
3. EDTA + EGTA: protease inhibitors, prevent oxidative damage (so divalent cations (Ca2+/Mg2+) can’t damage proteins)
4. Sodium fluoride and pyrophosphate: protease inhibitors
5. Triton X-100: detergent that helps solubilise proteins
6. Protease inhibitor: prevents breakdown of proteins
7. b-mercaptoethanol: reduces disulphide bridges and loosens out proteins

Everything kept on ice to slow denaturation

Question 6

Bradford Assay

1. What were the lysates diluted to?
2. How was this assay used to determine protein conc?
3. Why was this assay performed?

Answer 6

1. Lysates diluted 1:10 and 1:50
2. Used to determine protein concentration in unknown sample (used more commonly than Lowry assay). Looked at absorbance and then multiplied by dilution factor to determine protein concentration
3. This lets you know what volume of protein to add to get 10mcg

Question 7

PCR

1. What is PCR?
2. What gene was used and what was the difference between both samples?
3. What were the 3 different kinds of primers used?
4. What machine was used to perform PCR?
5. What was the gel through which DNA was separated?
6. What was Sybr-safe used for?
7. What did the denaturation step involve?
8. What did the annealing step involve
9. What did the extension step involve?

Answer 7

1. Fast, inexpensive method of molecular photocopying (amplifying DNA). At the end of each cycle the strands of DNA doubles and this is repeated approx. 30 cycles
2. K-Ras used from mouse to see if it was WT (homozygous) or mutated (heterozygous)
3. Primers: one forward primer starting at 5’ (Primer 1 binds to both WT and M), two reverse primers starting at 3’ (primer 2 only binds to WT; primer 3 only binds to M)
4. Performed in thermocycler with specific program
5. Separated with agarose gel as biggest bits of DNA move the shortest distance
6. Sybr safe: binds to double-strand DNA and fluoresces under UV light
7. Denaturation: heated up to 95 degrees
8. Annealing: cooled to 61 degrees and primers anneal to complementary strands
9. Extension: heated back up to 72 degrees (master mix: dNTPs, MgCl2 and Primers; thermophilic Taq polymerase can work at high temperatures)

Question 8

Western Blot Day I

1. What conc of sample buffer were samples made with?
2. What kind of gel was used in the electrophoresis tanks?
3. What was the solid surface that the proteins were transferred onto?
4. What stain was used to visualise bands on the solid surface?
5. How were the proteins de-stained?
6. How were proteins that weren’t of interest blocked from binding?
7. What were the primary antibodies used?

Answer 8

1. Samples created using lysates and 4X sample buffer
2. Acrylamide gels used in tanks and protein runs towards positive side (as it is -ve). Ladder used to separate sizes of protein for reference
3. Protein transferred onto solid nitrocellulose surface
4. Ponceau S used to visualise if bands were present on solid surface
5. Destained with TBST
6. Marvel non-fat milk used to bind to proteins, blocking those that weren’t of interest
7. Primary antibodies: anti-GFP and anti-GAPDH antibodies used to bind to proteins of interest

Question 9

Western Blot Day II

1. What were the secondary antibodies used?
2. What was the antibody combined with?
3. How did luminol cause light to be given off?
4. How long were samples exposed for?
5. What was GAPDH?
6. What was GFP?

Answer 9

1. Secondary antibodies (from rabbit): specifically bind to primary antibodies
2. Combined with horse radish peroxidase which gives light
3. Luminol oxidises HRP, causes it to become excited, HRP comes back down to ground state and gives off light
4. Ready to be exposed in dark room: first for 1 minute then for 5 minutes
5. GAPDH is housekeeping protein which should be in all cells (to check presence of protein and technique); should be uniform bands in all cells
6. GFP: should only be visible in cells that had undergone transfection (DNA samples and not in mock samples)

Question 10

What are the three formulae to remember for exams?

Answer 10

1. C1V1 = C2V2 (use for dilutions)
2. n = cv
3. n = m / GFM