Gel electrophoresis of formaldehyde psyllids
Gel electrophoresis of formaldehyde psyllids
Formaldehyde-agarose gel electrophoresis (formaldehyde-agarose gel electrophoresis) Formaldehyde is a commonly used denaturant for RNA. It is used for (1) RNA isolation and (2) RNA purification.
Operation method
Colloidal electrophoresis of formaldehyde psyllids
Principle
rRNA accounts for 80~85% of the total cellular RNA, after staining with ethidium bromide, the two main RNA ribbons on the colloid should be large and small rRNAs (28S and 18S for eukaryotes, 23S and 16S for prokaryotes); scattered near the small rRNAs, with faint smear, are mRNAs, this is because mRNAs do not exist in large quantities and vary in length. This is due to the fact that mRNAs are not present in large quantities and are of different lengths. The shorter 5S rRNAs and tRNAs also appear as specific color bands below the colloid.
Materials and Instruments
RNA Samples Move I. Preparation of experimental materials Caveat 1. This experiment is based on the method of Sambrook and Russell (2001). 2. Always wear gloves when performing this experiment. 3. formaldehyde and formamide are kept in an airing cupboard. Common Problems 1. For electrophoretic analysis of formaldehyde-containing psyllium colloids, formaldehyde-containing psyllium colloids must be prepared, and RNA must be denatured with formaldehyde and formamide to ensure that its dimeric structure is sufficiently opened. 2. 2. Because formaldehyde is a possible carcinogen, the preparation of the colloid and the electrophoretic analysis should be done carefully in an airing cupboard. 3. 3. In addition, formaldehyde-containing psyllium colloids are slippery and prone to breakage, and special care should be taken when moving the colloids. Since rRNAs make up 80-85% of the total cellular RNA, the two main RNA bands present on the colloid after staining with ethidium bromide should be large and small rRNAs (28S and 18S for eukaryotes, 23S and 16S for prokaryotes). 4. mRNAs are scattered around the small rRNAs and are faintly smeared because they are present in small quantities and are of different lengths. Shorter 5S rRNAs and tRNAs also appear as specific bands below the colloid. For more product details, please visit Aladdin Scientific website.
formaldehyde gel running buffer Formaldehyde gel loading buffer Ethidium bromide DEPC
Thermostat sinks Microcentrifuge Mupid II Mini electrophoresis tanks Gel casting apparatus UV transilluminator Protective masks Polaroid cameras Ventilation cabinets
1. Instruments
65℃ constant temperature water bath; microcentrifuge; Mupid II mini electrophoresis tank and glue casting apparatus; UV transilluminator; protective mask; Polaroid camera; air extraction cabinet.
2. Drugs and reagents
RNA (I-1, I-2, U-1, and U-2) extracted from the bacteriophage and RNA (#1, #2, #3, and #4) prepared from the extracellular transcription reaction of the positive and negative strands.5xformaldehyde gel running buffer (0.1 M MOPS, pH 7.0; 40 mM sodium acetate; 5 mM EDTA)Formaldehyde gel loading buffer (0.25% bromophenol blue; 0.25% xylene cyanol; 50% glycerol; 1 mM EDTA)Ethidium bromide (1 ug/uL in dH2O/DEPC)dH2O/DEPC
II. Method Steps1. Prepare a piece of 1.2% formaldehyde psyllium colloid
(1) Weigh 0.48 g agarose in a 100 mL serum vial and add 25 mL of dH2O/DEPC.
(2) After dissolving the agarose by microwave heating, shake the serum vial slightly to make the mixture homogeneous, and then transfer it to 60℃ constant temperature water bath for 5 min.
(3) Transfer the serum vial with agarose solution to a fume hood, add 8 mL of 5x formaldehyde gel running buffer and 7.2 mL of formaldehyde, and gently shake the vial to mix well.
The total volume of the gel is 40 mL. In order to avoid rapid coagulation of the agarose solution due to the rapid drop in temperature, it is important to move quickly, but avoid violent shaking, which may create a large number of air bubbles that could interfere with the coagulation of the gel.
(4) Pour the mixture as quickly as possible into the colloid mold in the fume hood and insert a teeth comb. Allow at least 30 minutes for the colloid to solidify.
2. Electrophoresis
(1) To perform electrophoresis, place the solidified parsley gel into the electrophoresis tank and add the appropriate amount of 1x formadehyde gel running buffer.
(2) Pre-run the gel at a constant voltage of 50 V for 5 min.
(3) Inject the 8 RNA samples listed above in order and electrophoresis at a constant voltage of 50 V. When the tracking dye moves to two thirds of the gel, turn off the power and move the gel to the UV transilluminator box to observe the presence of the RNA bands and take pictures for evidence.
Note: This piece of colloid will be used for northern transfer experiments, so please handle it carefully!