Calculate the percent agarose in the gel solution Molecular Techniques Laborator

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Calculate the percent agarose in the gel solution

Molecular Techniques Laboratory Lab 2: Agarose Gel Analysis of PCR Product; RE Digest PCR Product & Vect 1. Set up a 3 restriction enzyme digests of the DNAs provided in 1.5 mL microfuge tubes accord Agarose Gel Analyses of RE Digested PCR Product & Vector Table 2-1. The instructor will tell you which enzymes you need to u The reaction in Tube 3 is an uncut plasmid control. Do not add enzyme to this tube It is up to you to calculate the volumes of DNA, 10X buffer, water and enzyme needed. A make sure you select the correct 10X buffer that is compatible with both enzymes. Rer to record all of these values in your notebook! a. Table 2-1 Restriction Enzyme Digests Enz-2 Water Total DNA (ug) Vol 10X Vol Enz-1 (uL) Buffer (HL) 10 units_ -10 units(uL)--Vol (AL Tube | PCR product 50 50 15 (from Tube l) 30 | 1.0 g pMAL 3 100 ng pMAL none none 2. Incubate the reactions at 37°C for 1 hour 3. Place the remaining, undigested PCR product and pMAL vector at-20°C. You WILL NEEL 4. 5. them in future labs! At the end of the digestion, heat kill the reactions by incubation at 65°C for 15 min Place samples on ice Part III: Agarose Gel Electrophoresis of Digested PCR Product and Vector Materials Agarose gel and electrophoresis buffer from Part I 5 RE digested PCR product from Part II 5 RE digested pMAL-c5X from Part II 6X loading dye 15 L of uncut pMAL-cSX from Part II DNA size standards Load 5 L of the MW ladder, 5 from Tube l' 5 from. Tube 2 and the entire 15 from Tube into unused wells of the gel from Part I 1. Remember to mix the samples from Tubes l-3 with 2 L of 6X loading dye. a. 2. Connect the leads from the gel box to the power supply. ALWAYS electrophorese the samples towa the positive (red) electrode. 3. Run the gel at 90 V for 30 minutes 4. PLACE THE REMAINING DIGESTED DNA FROM TUBES 1 & 2 at -20°C! They will be used in Lab 3. 5. At the end of the run, look at the gel using the UV transilluminator and take a picture. NOTE:B SURE THAT THE UV SHIELD IS IN PLACE WHILE LOOKING AT THE GEL. Compare this to the photo of the gel from Part I. Is the PCR product still the same size b. Compare the patter n of the bands for the cut and uncut pMAL. Does pMAL appear to be expected size? pare the intensities of the digested PCR product and pMAL vector. How much p ? Use this value to estimate the amount of PCR product. The inte MA c. Com was loaded on the gel of a DNA stained with GelGreenTM is proportional to the mass of DNA in the band. 6. Dispose of the agarose gel and electrophoresis bu fer in the appropriate locations.

Explanation / Answer

Agarose concentration is taken in percent values depends upon the requirement in gel.

if you want 1% agarose gel then we have to dissolve 1 gram of agarose in 100 ml of buffer.

If we wants to make 0.5% agarose gel then we have to desolve 0.5 gram of agarose in 100 ml of buffer.

when we increase the percent amount of agarose than the pore size of gel is reduced it is applicable for the separation of small molecules.

when we use least concentration of agarose then we have to add very minute quantity of agarose to make agarose solution. in this condition gel have large pore size.

Pore size of gel is inversely proportional to agarose concentration.

For desired concentration we have to add desired amount of agoras in 100 ml of buffer.

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