Summary of Techniques

Question 1

Describe the important reagents for PCR

Answer 1

• Template
• dNTPs
• Taq pol
• Primers (forward & reverse)
• RT-PCR: SYBR green

Question 2

Describe the important reagents for ELISA

Answer 2

• Capture Ab against antigen
• Biotinylated Ab against antigen
• Streptavidin conjugated to HRP
• Substrate
• Samples (potentially with the antigen)

Question 3

Describe the important reagents for FACS

Answer 3

• FACS buffer:
  
  • FCS: nutrients for cells
  • ​Sodium azide: prevents internalisation of Ab
  • EDTA: prevents cells clumping
  • PBS: prevents osmotic rupture
• Fluorophore conjugated Abs
• Cells

Question 4

Describe the important reagents for Immunohistochemistry

Answer 4

• Paraffin embedded tissue cut into slices
• FCS: blocking of sample
• Isotype control (rat IgG against irrelevant Ag)
• Rat Abs against antigens of interest
• HRP conjugated detection Abs (against Rat IgG)
• Substrate: H₂0₂, DAB
• Mayer’s haemotoxylin stain

Question 5

Describe the control for Immunohistochemistry

Answer 5

• Blocking
  
  • ​With FCS
  • Prevents detection Abs from binding all over the slide
• Isotype control
  
  • ​Rat IgG against irrelevant Ag
  • Indicates if there is non-specific binding occuring

Question 6

Describe the control for FACS

Answer 6

• Gate on population known to be negative
• NB compensation needs to be performed

Question 7

Describe compensation in FACS

Why does it need to be done?

How is it performed?

Answer 7

• Remember, in FACS, a variety of fluorophores will be used (conjugated to differen detection Abs)
• These fluorophores will most likely have overlapping emission spectra
• Hence, if there is no compensation, there is the potential for false positive
  
  • ​eg PE spetrum leaks into FITC spectrum. A PE only positive cell may appear double positive
• Compensation is performed by subtracting the overlapping, ‘false positive’ from the true positive
• This is determined by running a single colour control (ie a cell suspension stained with only one set of Abs) through the flow cytometer

Question 8

Describe the control for PCR

Answer 8

• Non template control
• Allows one to see whether there has been any contamination (DNA contamination) of the wells
• Generation of a product in the NTC means that there has probably been contamination of some sort and that the other results are not valid

Question 9

Describe the control for ELISA

Answer 9

• Wells are left untreated with the sample
• Indicates whether there is any contamination of the wells