For our experiments to be rightly executed, some standard experimental procedures were followed. To achieve our goals of detecting HPV16 E7 gene, we had to test out multiple protocols and alter them to suit our needs. To ensure reproducibility, all of our reagent compositions and exact procedures were carefully noted and are presented in detail in the following tabs:

• The first thing to be done in the lab was to prepare all of our reagents in the desired concentrations. Most of the oligos were obtained in the form of lyophilized powders. The concentrations, volumes, buffers and methods used by us are described in great detail:
  

  STOCK SOLUTION PREPARATION
  

• The next step was to amplify our target gene i.e. E7. This was done so as to increase the available concentration of E7 to carry out a large number of replicates. But prior to the amplification, primers for E7 were designed, ordered and prepared to be used in the PCR reaction
  

  PRIMER PREPARATION
  

  PCR FOR E7 AMPLIFICATION
  

• The amplified E7 was then subjected to agarose gel electrophoresis to check whether E7 was correctly amplified.
  

  AGAROSE GEL ELECTROPHORESIS
  

• E7 then had to be eluted from the gel to obtain it in solution form to be used for CRISPR reaction
  

  DNA ELUTION
  

• The concentration of the DNA elute was figured out using a Nanodrop in the following way:
  

  NANODROP FOR CONCENTRATION DETERMINATION
  

• The first protocol was tested out using the NEB LbCas12a enzyme. The protocol suggested by NEB [1] was followed in the following manner:
  

  CRISPR/Cas12a REACTION SYSTEM PROTOCOL #1
  

• The results obtained from the first protocol proved to be unsuccessful, thus the protocol was altered according to the observations and inferences from protocol 1 (Read more about observations in the Results page under the Wetlab tab).
  

  CRISPR/Cas12a REACTION SYSTEM PROTOCOL #2
  

• Ideal results were initially produced after following the second protocol. However, they were not reproducible. LbCas12a from IDT [2] was tested out to eliminate the possibility of lack of compatibility of the guide RNAs with the enzyme ordered from NEB.
  

  CRISPR/Cas12a REACTION SYSTEM PROTOCOL #3
  

• Unsuccessful replicates were observed after following three protocols. In the urgent need for obtaining accurate results, literature was reviewed and multiple discussions were held with our PI and his graduate students and the potential problems with the previously followed protocols were figured out and a new protocol was tested out after debugging.
  

  Debugging
  

  CRISPR/Cas12a REACTION SYSTEM PROTOCOL #4
  

• The results from Protocol 4 were encouraging however hopeless simply due to the fact that the result depended on all of the ssDNA reporter to be cut which is not feasible. The problem was once again analysed and the protocol was completely changed. The expected results also changed with the same.The most logical way to tackle the problem at hand was to increase the reporter concentration so much so that we can eliminate the control line in the case of a negative reaction. This would happen because the reporter would be enough to bind to all of the Anti-FITC antibodies on the conjugate pad and none would remain to go to the Control Line.This new protocol gave accurate and reproducible results that were replicated 12 times with three different guide RNAsThe new protocol also worked with room temperature incubation by changing the incubation time from 2.5 hours to 4 hours. Exact details of the protocol can be found here:
  

  CRISPR/Cas12a REACTION SYSTEM PROTOCOL #5