Importance Of Restriction Digestion Analysis Of Dna

Restriction Digestion Analysis of DNA

Aim

• To preform restriction digest on a plasmid pEX-A00446-M03.

• To estimate using a plasmid map the size of the plasmids.

• To compare the DNA bands on the gel to known DNA ladders.

Introduction

Restriction enzymes are bacterial proteins that act as a defence in these organisms. These enzymes search for a certain sequence of bases that they can relate to in a DNA strand. Once found, the restriction enzymes attach to the DNA molecule and cut the strands of a double helix. This happens along the full length of DNA until it breaks up into many smaller pieces. These smaller pieces are known as fragments and their sizes are measured in base pairs or kilobase pairs. As the sequence of base pairs for

Method

As per molecular biology manual 2015 but the following changes did occur.

Setting up of Restriction Digest Restriction

Enzyme Buffer

Xho I D

Sal I D

Sac I Y

Double Digest – Xho I and Sac I DD

• The following restriction enzymes and buffers were used:

• The samples were incubated at 37°C for 3 days

Analysis of Digested Fragments

• Load both the 1kb DNA ladder and the 100bp DNA ladder and the digest samples to the gel.

• Run the gel for 45-50 minutes at 130V

• View gel under UV transilluminator, record data and take photo of gel for further analysis of the DNA bands.

Results

Calculations Week 1

Reagent Final Conc required Single Digest Double Digest

10 X Buffer 1X 1ul 1ul

DNA 4mg/ml 0.4ug/ul 1ul 1ul

RE 1 (10U/ul) 10U 1ul 1ul

RE 1 (10U/ul) 10U - 1ul

Sterile H20 - 7ul 6ul

Total Volume 10ul 10ul

Figure 1: Table showing the volumes required for each reagent

All the above calculations were calculated by a 1 in 10 dilution and then making the sample up to 10ul with sterile H20.0

Prediction of Enzymes- Schematics

Xho 1

Sac 1

Sal 1

Xho 1 and Sac