Electrophoresis, transfilm

related topicsTransfilm is a prerequisite for the transfer of
DNA
from
agar
sugar gels to solid-phase supports (usually nylon membranes) and is a prerequisite for various follow-up studies (e.g. RFLP analysis, screening verification of positive cloning, etc.) involving molecular hybridization.experimental purpose: master the principle of South blotting and operating steps
Experimental principle:
1. The way of the transfilm:
upward capillary transfer
downward capillary transfer
simultaneous transfer to two membranes
electric transfer
vacuum transfer
2. Types and options of solid-phase support:
fiber pigment membrane: non-co-priced binding, brittle, fragile, loss-prone DNA, < 500 bp of DNA invalid, pre-membrane work (from improving transfilm efficiency, benefiting after transfilm use, etc.)
nylon membrane (positively charged) high strength, non-destructive, with a large DNA binding capacity, it can absorb denatured DNA,
nucleic acid
irreversibly combined with the nylon membrane in a co-priced binding manner. Nylon membrane on both sides have the same adsorption of DNA function, no matter which side can be durable, can be repeatedly used more than 10 times (10-20 times), through capillary tube (capillary adsorption) action, the DNA from the gel to the membrane. DNA transfer to the membrane is a tape type on the replication glue, which can be fixed in a vacuum
80-100
2-4 hrs.
3. Options for transfer buffers:
positively charged nylon membranes: high salt ion strength (SSC) available, but not full membrane potential;
nitric acid fiber membrane: high salt ion strength promotes DNA-membrane binding (20×SSC), low salt ion strength causes small fragments of DNA to be lost during metastasis, pH>9, DNA can not bind to membrane.
4. The duration of transfer is approximately 12 hrsefficient, but there is no remedy, so every step should be strictly operated.
experimental materials and
reagents
: enzyme digested DNA samples, nylon membrane, 0.2N HCl, 0.4N NaOH, etc.
experimental steps:
1. 0.8% agar electrophoresis
glue: pay attention to the quality of agar sugar, the concentration of gum, thickness (<5mm) and ionism. General large electrophoresis tank preparation 250ml 0.8% agarose gel, using a 42-hole comb (economical, efficient)
sample, sample: DNA samples indicated a slightly more dose
electrophoresis: general 1-1.5V/cm voltage, To allow DNA to migrate to an appropriate distance, the general indicator moves about 10-11cm (large
electrophoresis tank
:40V×12-15hrs, small electrophoresis slot 30V×4-5 hrs)
2. Pre-film preparation:
according to the size of each gel prepared two slightly larger than the glue
filter paper
(11×12.5 cm), two filter paper used as a salt bridge, one with The same size nylon film (10×10.5cm), two glass discs, two
organic
glass plates, a glass rod, a stack of absorbent paper slightly larger than the nylon film, etc.
3. Add a sufficient amount of 0.4N NaOH to a glass plate, place the washed glass plate, and make a salt bridge as shown (operation demonstration).
4. Pre-treatment of the gel:
a) Remove the gel from the electrophoresis tank and place it on a plastic plate, cut the glue into the appropriate size with a cutting plate, cut off the upper right corner (front end of the last sample) as the electrophoresis direction mark
b) Turn the gel over and place it in a glass plate with a sufficient amount of 0.2N HCl, gently shaking 10min to make the indication Until the agent becomes yellow (dehydrated)
c) pours out the HCl solution, adds distilled water rinse gel
d) pours the distilled water, adds 0.4N NaOH medium and
e) and sprinkles 0.4 NaOH on the salt bridge filter paper, immediately places the glue on the salt bridge (bubble)
5. The plastic sheet around the glue is closely connected to the glue to prevent a short circuit (water-absorbing paper is connected to the salt bridge)
6. Pour a sufficient amount of 0.4N NaOH on the surface, carefully place the film (pre-wet 0.4N NaOH) so that the film covers the entire piece of glue (requires one success and cannot be moved)
7. Put 2 sheets of filter paper on the membrane, the filter paper size is 15×12cm
8. Put not less than 5cm thick absorbent paper, put on a glass plate, its pressure about 500g of heavy objects, transfilm 12 hrs
9. The film is finished, with 2 × SSC rinsing film twice, each for five minutes. Use EB dyeing glue to detect transfer effects.
10. Wrap the film in two sheets of filter paper and place it in a vacuum
-drying box
80-100 degrees C, drying 2-4 hrs.