The overall goal of this procedure is to isolate PCR ready nucleic acids and immunoreactive protein from a sample without the need for electricity or laboratory infrastructure. Isolation of nucleic acids in proteins begins with mixing the sample with guine and thiocyanate to lice the sample and passing the mixture over the sorbent in the extraction pipette. Five times after the fifth and final passage over the sorbent place the mixture into protein extraction, buffer, and set aside for isolating the immunoreactive protein.
Next, the sorbent and bound nucleic acids are washed by passing 95%ethanol over the sorbent. Numerous times the sorbent and bound nucleic acids are then dried by passing ambient air over the sorbent for five minutes. The final step for PCR ready nucleic acid isolation is to allude the nucleic acids from the sorbent using 250 microliters of tris or another similar aqueous solution for isolation of the immunoreactive protein.
After mixing the sample and protein extraction buffer, use a second extraction pipette and pass the mixture over the sorbent 15 times. The next step is to wash the sorbent with bound protein by passing 95%ethanol over the sorbent numerous times. Then the sorbent and bound protein are dried by passing ambient air over the sorbent for five minutes.
The final step of the protocol is to elute the protein from the sorbent. Ultimately, the extraction pipette used in this procedure allows for the electricity free isolation of PCR ready nucleic acids and immunoreactive protein. Hi, my name is Dave Pawlowski.
I'm a senior research scientist at Kubrick Incorporated in Buffalo, New York, and we'll be learning a technique today that will isolate both protein and nucleic acids in line without the need for a centrifuge or a vortex or any other extraneous lab equipment Prior to sample lysis. Ensure samples are in liquid form. If sample is solid, for example, food or stool, the sample should be suspended in a liquid media such as water or phosphate buffered saline.
Mix 500 microliters of the sample with 500 microliters of six molar guine thiocyanate in a 1.5 milliliter tube. Next, depress the bulb of an extraction pipette, expelling the ambient air. Pull the entire one milliliter sample into the extraction pipette over the absorbent material by allowing the bulb to slowly expand.
Invert the extraction tool such that the sorbent beads and sample drain into the bulb. Grind the sorbent beads with the sample in the bulb. Being careful not to expel the sample out of the extraction pipette.
Invert the extraction pipette, and expel the sample into the original 1.5 milliliter tube. Pull the entire one milliliter sample into the extraction pipette, passing over the sorbent at a moderate rate. Being careful to keep the sorbent in the neck of the extraction pipette, expel the entire sample into the 1.5 milliliter tube by depressing the bulb.
Repeat this process, passing the sample over the sorbent four more times for a total of five passes. After the fifth pass over the sorbent, expel the entire one milliliter sample into four milliliters of the protein extraction buffer in a 15 milliliter conical tube. Mix the tube by inversion, then close the tube and set it aside for later.Use.
Wash the isolated nucleic acids by passing one milliliter of 95%ethanol over the sorbent three times, returning the ethanol to its original tube. Upon final passage, repeat the wash three times using a second milliliter of 95%ethanol wash. The next step of the procedure is to dry the sorbent nucleic acid recovery is best done when the sorbent is dry.
I know this step is tedious. However, if you complete this step in the recommended timeframe, your results will be better. Press and release the bulb for five minutes.
Passing ambient air throughout the extraction pipette to dry the nucleic acids and sorbent bed, wipe residual ethanol from the tip of the extraction pipette. Using a Kim wipe, recover the nucleic acids by passing 250 microliters of 10 millimolar triss over the sorbent five times. The triss buffer can be replaced by any other suitable aqueous solution such as PBS.
The resulting solution contains the extracted nucleic acids to perform protein extraction first, mix the sample in protein extraction buffer that was previously set aside by inverting the tube pass approximately two to three milliliters of this five milliliter sample over the sorbent of a new extraction. Pipette expel the entire sample into the same 15 milliliter conical tube. Being careful to keep the sorbent in the neck of the extraction pipette.
Repeat this process. Passing the sample over the sorbent 15 times. Wash the sorbent and the bound protein by passing one milliliter of 95%Ethanol over the sorbent bed three times, ejecting the ethanol into its original tube wash three more times using a second milliliter of 95%ethanol wash.
The next step of the procedure is to dry the sorbent again. As with nucleic acids, protein recovery is best done when the sorbent is dried. For a longer period of time.
Press and release the bulb for five minutes, passing ambient air throughout the extraction pipette. To dry the protein sorbent bed, wipe the residual ethanol from the tip of the extraction pipette. Using a Kim wipe, recover the protein by passing 250 microliters of PBS over the sorbent five times.
The PBS buffer can be replaced by any other suitable aqueous solution, such as 10 millimolar tris, pH 6.8. The resulting solution contains the protein content of the sample. The shiga toxin genes associated with entero hemorrhagic e coli oh 1 5 7 H seven strain E DL 9 33 were amplified from a nucleic acid protein extraction procedure using the extraction pipette.
The shiga toxin genes, Shiga toxin one and shiga toxin two are found on temperate lambda like bacteria phage and defective bacteria phage under the control of the PR promoter. The activation of the SOS response in e coli leads to pro phage in induction and or toxin production. In this case, there is a large increase in the amplification of the sugar toxin two gene when the SOS response is induced by mitomycin C in comparison to the untreated control.
This is suggestive of pro phage induction and phage genome replication. The recovered protein from the nucleic acid protein isolation experiments described in the video is immunoreactive. The isolated protein fraction was applied to the rapid check e coli O 1 57 lateral flow assay strips.
Controls show a positive identification in all samples harboring e coli oh 1 5 7 with or without shiga toin induction as visualized by the presence of two red bands. The data also show positive results for those samples harboring e coli oh 1 5 7 that were processed for nucleic acids and protein using the extraction pipette. These data show that the isolated protein from each sample is immunoactive, thus triggering a positive response.
The isolated protein is suitable for electrophoresis as depicted here in a kumasi stained 8%Acrylamide gel loaded with BSA isolated from six molar gudina isothiocyanate. Using the extraction, pipette and associated protocol, the electrophoretic mobility of the isolated protein is identical to that of the control samples. Once mastered, you can perform this technique in under 20 minutes.
After watching this video, you should have a clear understanding of how to isolate proteins and nucleic acids from the simple device. Thanks for taking the time to watch our video.
