Practicals learnt and performed:
1. Starch Isolation
2. PCR amplification in parents using various primers
3. Standard curve preparation of potato amylose
4. Check for working of primers in vector PCA(pCambia)
Standard curve preparation:
The basic principle used is, the starch is made to react with iodine which forms a colored complex (violet). The intensity of color is estimated using Spectrophotometry and the absorption is noted. A graph is plotted using the data obtained and the result is analyzed.
Checking for working of primer in Vector PCA(pCambia):
This experiment was performed to check whether the particular primers( forward and reverse) that were designed could amplify the region intended in PCA. A reaction mixture comprising Master Mix (includes Taq polymerase, Buffer, MgCl2, dNTPs), Primers (both forward and reverse), template(Vector PCA), and Water was prepared. The reaction was carried out in PCR thermo cycler under specific reaction parameters. The results were analyzed by performing gel electrophoresis to check if the primer could amplify the desired region of the vector.
Today, I prepared 1% Agarose gel properly and rectified the mistake I did yesterday. Yesterday probably, I did not heat the agarose gel solution for appropriate time(though the solution looked clear) and did not ensure that all the bubbles were removed.
1. Starch Isolation
2. PCR amplification in parents using various primers
3. Standard curve preparation of potato amylose
4. Check for working of primers in vector PCA(pCambia)
Standard curve preparation:
The basic principle used is, the starch is made to react with iodine which forms a colored complex (violet). The intensity of color is estimated using Spectrophotometry and the absorption is noted. A graph is plotted using the data obtained and the result is analyzed.
Checking for working of primer in Vector PCA(pCambia):
This experiment was performed to check whether the particular primers( forward and reverse) that were designed could amplify the region intended in PCA. A reaction mixture comprising Master Mix (includes Taq polymerase, Buffer, MgCl2, dNTPs), Primers (both forward and reverse), template(Vector PCA), and Water was prepared. The reaction was carried out in PCR thermo cycler under specific reaction parameters. The results were analyzed by performing gel electrophoresis to check if the primer could amplify the desired region of the vector.
Today, I prepared 1% Agarose gel properly and rectified the mistake I did yesterday. Yesterday probably, I did not heat the agarose gel solution for appropriate time(though the solution looked clear) and did not ensure that all the bubbles were removed.
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