TITLE: Gradient PCR 
AUTHORS: Ashwinuday 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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.</div></div>

[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  and 5 test samples were made for the 5 different annealing temperatures to be vaired: -  Itemsnegative controltest sample (*5)ultrapure water (from Milli-Q)40 ul39 ulpfu 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 polymerase1 ul1 ulTemplate (100ng/ul)1 ul (100ng)1 ul (100ng)
50 µl
Itemsnegative controltest sample (*5)ultrapure water (from Milli-Q)40 ul39 ulpfu 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 polymerase1 ul1 ulTemplate (100ng/ul)1 ul (100ng)1 ul (100ng)
?. Perform PCR using a Thermo cycler with the following temperature settings (All temperatures are in degree Celcius).r In the thermo cycler gradientPCR option is kept and the following gradient is specified. Then the 5 test samples are labelled based on their temperature and kept in the correct column in the thermo cycler.TemperatureTime955 min9530 secs51, 53, 55, 57, 5940 secs722 min725 minSet the 2nd, 3rd and 4th steps to repeat 30 times (or maximum till 35)
TemperatureTime955 min9530 secs51, 53, 55, 57, 5940 secs722 min725 min
The PCR product can be immediately run analyzed using gel electrophoresis or can be stored in   or lower to be analyzed later.
?. To prepare the 1% agarose gel, mix  of agarose in  of 1X TAE buffer and heat well to form a clear solution.
All items which interact with the gel and EtBr should be handled with gloves. Also ensure, the gloved hands are not used to touch anything other than gel materials.
0.5 g
50 mL
Based on requirement higher amounts of gel can be prepared. So, accordingly larger combs and higher amount of EtBr can be used.
?. Then the mix is allowed to cool till it is bearable to touch. Carefully add  of EtBr to the mix and swirl well.
2 µl
?. 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.
If there is any bubble remove it with a tip soon after pouring
?. 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  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
10 µl
?. 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
?. Check bands corresponding to samples from which temperature shows good intensity and is at the right size and choose that annealing temperature for further PCRs