Microbial diagnostics – UTI labs
Use your own language to write the steps and also summarize them briefly. ( write only the important informations).
Primer design
FORWARD
5’ CAC GAT CAG ATA AAC TCC AGT GGG ‘3
MELT TEMP 57.1 ºC
GC CONTENT 50%
Reverse
5’ CCA TCG AGT TCA TGT TGT TAT CCG ‘3
MELT TEMP 55.8 ºC
GC CONTENT 45.8 %
- Set up PCRs using primers designed in
You will use PCR to differentiate between UPEC and non-pathogenic E. coli.
Working individually, you are provided with a rack of PCR tubes, P2 and P10 pipettes and tips, Mango PCR mix (contains DNA polymerase, dNTPs, buffer and gel loading dye), PCR primers, PCR grade water and genomic DNA isolated from UPEC and non-pathogenic E. coli. The strains we are using are CFT073 (UPEC) and MG1655 (non-pathogenic E. coli).
You will test your own PCR primers to see if these can differentiate between UPEC and non-pathogenic E. coli. In addition, you are provided with a pair of UPEC control primers (these detect UPEC but not non-pathogenic E. coli) and a pair of E. coli control primers (these detect both UPEC and non-pathogenic E. coli).
You will test each pair of primers using UPEC and non-pathogenic gDNA as template. To check that your reagents are not contaminated, you will also set up no template control reactions without any gDNA template. These should not yield any PCR product.
Label all of your tubes (including your name and bench letter) and assemble the PCR reactions on ice according to the guidance below. Wear gloves and change your pipette tip after every step to prevent contamination.
UPEC PCRs:
In an Eppendorf tube, prepare a UPEC PCR mastermix comprising:
- 35 µl Mango Mix
- 28 µl water
- 3 µl of UPEC CFT073 gDNA
Aliquot 19 µl of this mastermix into each of 3 labelled PCR tubes then add 0.5 µl of each 25 µM primer according to the following table:
| PCR tube label | Forward primer | Reverse primer |
| UPEC pTest | Your forward primer | Your reverse primer |
| UPEC pUPEC | Control UPEC-F | Control UPEC-R |
| UPEC pEc | Control Ec-F | Control Ec-R |
Non-pathogenic E. coli PCRs:
In an Eppendorf tube, prepare a non-pathogenic E. coli PCR mastermix comprising:
- 35 µl Mango Mix
- 28 µl water
- 3 µl of non-pathogenic coli MG1655 gDNA
Aliquot 19 µl of this mastermix into each of 3 labelled PCR tubes then add 0.5 µl of each 25 µM primer according to the following table:
| PCR tube label | Forward primer | Reverse primer |
| Ec pTest | Your forward primer | Your reverse primer |
| Ec pUPEC | Control UPEC-F | Control UPEC-R |
| Ec pEc | Control Ec-F | Control Ec-R |
No template control PCRs:
In an Eppendorf tube, prepare a no template control PCR mastermix comprising:
- 35 µl Mango Mix
- 31 µl water
Aliquot 19 µl of this mastermix into each of 3 labelled PCR tubes then add 0.5 µl of each 25 µM primer according to the following table:
| PCR tube label | Forward primer | Reverse primer |
| NTC pTest | Your forward primer | Your reverse primer |
| NTC pUPEC | Control UPEC-F | Control UPEC-R |
| NTC pEc | Control Ec-F | Control Ec-R |
Place your 9 labelled PCR reaction tubes into a PCR machine. The PCR reaction will be run using the following conditions then stored in the freezer until next week:
1 cycle of 95 C 90 s
35 cycles of 95 C 30 s, 52 C 60 s, 72 C 90 s
1 cycle of 72 C 5 min.
- Agarose gel electrophoresis of PCR reactions.
You are provided with equipment for agarose gel electrophoresis, P10 pipettes and tips, a DNA ladder and your PCR reactions from last week.
Wear gloves. Load 10 µl of each of your PCR reactions into the wells of the agarose gel, alongside 5 µl of 1 kb DNA ladder. Make sure that you record which PCR reaction you have loaded into which well. Ask a lecturer/demonstrator for assistance to start the electrophoresis run.
Proceed with the other experiments. After 45 minutes, return to your gel and ask for assistance to visualise the DNA bands. Use your tablet to record the results, then answer the following questions:
- Are the no template control PCRs clean (no products)?
- Did the control coli primers detect both strains?
- Did the control UPEC primers detect UPEC only?
- Did your test primers detect UPEC only?
- If you have any unexpected results, can you think of any possible explanations for these?
p(12)
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