A series of concentration and volume control experiments were set up.

OBSERVATIONS FOR VERSION #1
100uL was a lot of fluid for the strip to absorb. The quantity of the solution needed reduction.

• In the first case, a very strong control line developed indicating that the strips worked perfectly fine.
• In the second case, solid control and test ines were seen indicating that the reporter with the FAM and biotin was in order and worked as expected.
• In the last case, a very strong test line and a weak control line was observed. The control line intensity was weak because there was a lot of FAM to which the Anti-FITC antibodies on the conjugate pad could bind to. This conjugate went to the test line where biotin bound to Streptavidin. Due to this, there was very little concentration of the Anti-FITC was left to go to the control line. Hence, the weak intensity.
  
  

A second set of experiments with reduced concentrations was performed to figure out the optimum concentration for the reaction.

OBSERVATIONS FOR VERSION #2

• The solution was able to flow properly and reached the end of the strip almost instantly. A solid control was observed
• The observed result and speed of flow of the buffer were very similar to the first strip.
• A weak control line was observed but the solution could flow till the end of the strip.
  
• Weak control line was observed but the solution could not flow till the end indicating that 20uL volume for the ssDNA concentrated reporter is too less mostly because the viscosity of the reporter solution is greater than the buffer that was initially made to flow on the strip with the same volume.
  
• Extremely weak control line mostly because the total number of moles of FAM available for the Anti-FITC antibodies increased a lot and hardly anything must have been left to go to the control line.
  
  

The following image shows the result of the 6 reactions that were set up following the first protocol

OBSERVATION

Both test line and control line were observed in all of the cases. We were expecting only the control line in cases where all of the DNA reporter would be cleaved.

INFERENCE

• CRISPR reactions didnt work properly. We inferred and after literature review, it was realised that the incubation time was less. For all further experiments, the incubation time was decided to be increased to 2.5 hours.
• It was also thought that the enzyme was not enough to cleave all the reporter so the enzyme volume used was also increased for further experiments.
• A possibility was considered that the NEB enzyme was not compatible with the guide RNA ordered from IDT.
  

The following image shows the result from the first time the second protocol was followed. This was the first successful experiment.

OBSERVATION

A heavy intensity control line is observed. The intensity of the test line is very weak; almost negligible.

INFERENCE

• All of the reporter molecules were cleaved. Very few remained that gave a very weak test line. This is very close to the desired result.
• After this experiment, the protocol was standardized according to the above mentioned table and the same procedure was followed for all further replicates.

REPLICATE SET #1

The following image shows the result of the 8 reactions that were set up in accordance with the table mentioned along with Replicate#1 in the second protocol.

OBSERVATION

• A higher intensity test line can be seen on strip 1 and 2 due to the higher concentration of reporter used.
• A very low intensity test line is seen when E7 is not added. This is not in accordance with the expected results. Ideally, a very strong test line should have been observed in combination with a control line.
• No test line was observed in the case where reporter was not added (Strip 4)
• A weak intensity test line was observed when the system was incubated for only 1hr 10mins. This could mean that the reaction time was not sufficient for all of the reporter to be cleaved
• When the Cas12 enzyme was omitted from the system, a weak test line and a very solid control line was obtained. Again, this result is not clearly distinguishable from the positive result.
• Extremely weak test line was obtained when guide RNA 2 and 3 were used in the system.
  
  
  

INFERENCE

• This result agrees with the previous successful attempt. However, the negative controls also display a very weak test line making it very difficult for the user to accurately distinguish between a negative and a positive HPV case.
• There is very little reporter in the system which is why the test line in any case is weak.

REPLICATE SET #2

OBSERVATION

Here, the first strip is the positive reaction. The results are very different from the results obtained the first time this protocol was followed. Moreover, there is no significant difference between strip 1 and strip 2. Thus, in this reaction setup, there is no way to distinguish between a positive and negative reaction.

REPLICATE SET #3

Exact same replicate was carried out again with unsatisfactory results

OBSERVATION

Negative and Positive results are almost identical. The protocol needs to be altered yet again.

OBSERVATION

• All of the reporter molecules were cleaved. Very few remained that gave a very weak test line. This is very close to the desired result. After this experiment, the protocol was standardized according to the above mentioned table and the same procedure was followed for all further replicates.
• However, in order to differentiate between a positive and a negative result, all of the reporter needs to be cleaved.
  
  

INFERENCE

• The reporter concentration needs to be minimised in order to make sure that almost all of the reporter gets cleaved as a result of the trans cleavage activity of Cas12.
  
• In order to ensure that CRISPR reactions work properly, the concentration of E7 was also increased.
  

DEBUGGING PART #1

OBSERVATION

• The first strip here shows 2nM concentration of the reporter used. The second, third and fourth strip are the results of using 5nM, 10nM and 15nM respectively 
  
• A very weak test line is observed in the case of 2nM concentration of the reporter used. The remaining strips show a good signal at the test line.
  

INFERENCE

• The second strip shows a good test line and utilises the least reporter concentration. Hence, for protocol 4, the concentration of the DNA reporter was fixed to 5nM.
  

Here, the concentration of E7 was increased to almost two times the previous concentration and the concentration of the reporter was reduced to 5nM as a result of Debugging Part#1

OBSERVATION

• All of the reporter molecules were cleaved. Very few remained that gave a very weak test line. This is very close to the desired result. After this experiment, the protocol was standardized according to the above mentioned table and the same procedure was followed for all further replicates.
• However, in order to differentiate between a positive and a negative result, all of the reporter needs to be cleaved.
  
  

INFERENCE

• The reporter concentration needs to be minimised in order to make sure that almost all of the reporter gets cleaved as a result of the trans cleavage activity of Cas12.
  
• In order to ensure that CRISPR reactions work properly, the concentration of E7 was also increased.
  

DEBUGGING PART #1

OBSERVATION

• The first strip here shows 2nM concentration of the reporter used. The second, third and fourth strip are the results of using 5nM, 10nM and 15nM respectively 
  
• A very weak test line is observed in the case of 2nM concentration of the reporter used. The remaining strips show a good signal at the test line.
  

INFERENCE

• The second strip shows a good test line and utilises the least reporter concentration. Hence, for protocol 4, the concentration of the DNA reporter was fixed to 5nM.
  

OBSERVATION

• Here, the first two strips display the results of a positive HPV sample adn the last strip shows the results of a negative HPV sample
• There is a clear and significant difference between a negative and a positive sample.
  
  

INFERENCE

• The new protocol works perfectly as any reporter that gets cleaved goes to the control line and gives a signal there.
  

REPLICATE #1

OBSERVATION

• There is a very clear distinction between a negative HPV (Left Strip) and a Positive HPV (Right Strip)
  

INFERENCE

• The new reagent composition and procedure works well and could be successfully replicated twice.
  

REPLICATE #2

OBSERVATION

• A clear distinction between a negative HPV and a positive HPV is observed.
  
• The intensity of the control line remains constant even on changing the guide RNA.
  
  

INFERENCE

• The new reagent composition and procedure works well and could be successfully replicated twice.
  

REPLICATE #3

OBSERVATION

• Successful replicates can be observed in the case of Guide RNA 1 and Guide RNA 2
  
• Satisfactory results cannot be seen when the incubation time was reduced to one hour.
  
• There is no significant difference between a negative HPV and a positive HPV when the reaction was incubated at Room Temperature for 2.5 hours.
  
  

INFERENCE

• All three guide RNAs work perfectly well and the reaction could be reproduced successfully.
  
• 1 hour of incubation is not enough to achieve the desired result
  
• The incubation time needs to be increased to produce successful results at Room Temperature. Thus, for all further replicates, the intubation time was increased to 4 hours.
  

REPLICATE #2

OBSERVATION

• The fourth replicate of each guide RNA (strips 1 to 3), was successfully carried out thereby, giving us proof of concept. There is a significant difference between positive HPV results and negative HPV results.
  
• By reducing the E7 concentration, no significant control line could be seen.
  
  

INFERENCE

• E7 concentration plays an important role in the development of the control line and upon reduction of the concentration down to 30nM, there is no significant difference between a positive HPV and a negative control.
  

REPLICATE #2

OBSERVATION

• After 4 hour incubation, strong signals on the test line were observed in the case of all three guide RNAs
  
  

INFERENCE

• Once again it was observed that the intensities of the signal did not vary significantly with the choice of guide RNA
  
• It was confirmed that room temperature incubation of this reaction system requires a greater time to produce successful results.