Today, screening of parents with more primers had to be performed. The steps include:
1. Performing PCR using required primers.
2. Gel electrophoresis of the amplified products
3. Gel documentation
4. Analyzing the gel for polymorphism in the parents.
A 3% Agarose gel had to be prepared for the purpose. performed the following steps.
1. Weigh 3g Agarose.
2. Add Agarose powder in 100ml 0.5x TBE buffer in a beaker (usually more 20-30 ml more Buffer is used considering the evaporative loss of buffer when heated)
3. Heat the Agarose solution by keeping in an Oven for 6-7 mins or until there are no lumps and residues of Agarose. A clear solution should be obtained
4. Swirl the solution in the beaker to remove the emerging bubbles until no more bubbles are seen.
5. Add 2- 2.5 microlitres Ethidium Bromide and mix well.
6. Set the Gel Electrophoresis Assembly.
7. Slowly pour the Agarose solution into the assembly making sure there is no bubble formation.
8. Place the combs gently into the gel and allow it to solidify.
After following the above procedure, the amplified PCR products were run in the gel.
Everything looked good. The samples could be seen running on the gel too. But, when proceeded for gel documentation, the gel looked very bad. The DNA's movement was hindered in the gel also the bands looked wavy and blurred. Polymorphism in the parents couldn't be identified. That gave a little heart ache as the whole PCR procedure went in vain.
The possible reasons for such result could have been
- The Agarose concentration must have differed a little while weighing
- Some bubbles must have remained in the gel that hindered the movement of the DNA threads.
- There was a delay in loading the samples after the gel was ready.
(You may suggest any other possible reasons, so that I could be more cautious while doing next time)
It was definitely little dissappointing for not having been able to prepare a perfect gel.
Hoping for improvement.
1. Performing PCR using required primers.
2. Gel electrophoresis of the amplified products
3. Gel documentation
4. Analyzing the gel for polymorphism in the parents.
A 3% Agarose gel had to be prepared for the purpose. performed the following steps.
1. Weigh 3g Agarose.
2. Add Agarose powder in 100ml 0.5x TBE buffer in a beaker (usually more 20-30 ml more Buffer is used considering the evaporative loss of buffer when heated)
3. Heat the Agarose solution by keeping in an Oven for 6-7 mins or until there are no lumps and residues of Agarose. A clear solution should be obtained
4. Swirl the solution in the beaker to remove the emerging bubbles until no more bubbles are seen.
5. Add 2- 2.5 microlitres Ethidium Bromide and mix well.
6. Set the Gel Electrophoresis Assembly.
7. Slowly pour the Agarose solution into the assembly making sure there is no bubble formation.
8. Place the combs gently into the gel and allow it to solidify.
After following the above procedure, the amplified PCR products were run in the gel.
Everything looked good. The samples could be seen running on the gel too. But, when proceeded for gel documentation, the gel looked very bad. The DNA's movement was hindered in the gel also the bands looked wavy and blurred. Polymorphism in the parents couldn't be identified. That gave a little heart ache as the whole PCR procedure went in vain.
The possible reasons for such result could have been
- The Agarose concentration must have differed a little while weighing
- Some bubbles must have remained in the gel that hindered the movement of the DNA threads.
- There was a delay in loading the samples after the gel was ready.
(You may suggest any other possible reasons, so that I could be more cautious while doing next time)
It was definitely little dissappointing for not having been able to prepare a perfect gel.
Hoping for improvement.
