PCR and gel electropheresis
Ashwinuday
Abstract
This protocol can be used to confirm genes or DNA of interest from a template using PCR. Also to amplify required amount of genes to larger amounts.
Steps
Prepare the working stock solution of all the reagents required. If already prepared and stored, takeout from the refrigerator and thaw on ice.
Prepare the following mixes each of total 50µL : -
| A | B | C |
|---|---|---|
| Items | negative control | test sample |
| ultrapure water (from Milli-Q) | 40 ul | 39 ul |
| pfu Buffer (10X) | 5 ul (1X) | 5 ul (1X) |
| Forward primer (20uM) | 1 ul (0.4uM) | 1 ul (0.4uM) |
| Reverse primer (20uM) | 1 ul (0.4uM) | 1 ul (0.4uM) |
| dNTPs (2.5mM) | 2 ul (0.1mM) | 2 ul (0.1mM) |
| Pfu polymerase | 1 ul | 1 ul |
| Template (100ng/ul) | 1 ul (100ng) | - |
Perform PCR using a Thermo cycler with the following temperature settings (All temperatures are in degree Celcius) :
| A | B |
|---|---|
| Temperature | Time |
| 95 | 5 min |
| 95 | 30 secs |
| 55 | 40 secs |
| 72 | 2 min |
| 72 | 5 min |
Set the 2nd, 3rd and 4th steps to repeat 30 times (or maximum till 35)
To prepare the 1% agarose gel, mix 0.5g of agarose in 50mL of 1X TAE buffer and heat well to form a clear solution.
Then the mix is allowed to cool till it is bearable to touch. Carefully add 2µL of EtBr to the mix and swirl well.
Then into a set gel cast with the appropriate comb (which is decided by the number of wells and the size of the wells required), pour the mix without bubbles.
Once the gel solidifies, carefully take it out with a gloved hand and place in the buffer tank with enough buffer to cover the immersed gel and remove the comb without breaking the wells.
Mix approximately 10µL samples with 1X loading dye and load into each gel. Load a 1kb ladder (or any suitable ladder) into the first lane. Run the gel at 100V
After the dye front crosses 75%of the gel length, stop the current.
Take the gel out (with gloved hands) by letting all the buffer out into the tank and then image the gel on UV.
Compare the sample bands with the ladder to estimate the bands of interest
