Question

A. Your lab To see if you understand what you did in our lab, answer the following based in the procedures for the restriction digest and the gel electrophoresis. 1. Using the PGLO map on p7 of the gel electrophoresis procedure, predict the size of the fragments generated by each enzyme EcoRI Hindill, and Pst! (the sizes you would expect to see on the gel.) (6 pts) Hindill -8 fragments were produced by the restriction enzyme. 2. Answer the calculation on page 5: how much agarose do you need to make 80 mL of 1% agarose gel? (2 pts) To cast your gel you will determine the amount of agarose to use. You will use a 1%TBE agarose gel. 0.8 gram of agarose needed for 80mL. 8. The video B. The video Answer the following to video from the Dolan DNA center on restriction digest/gel electrophoresis. Also there is a lot of good background on transformation, restriction enzymes and gels that could help for your quiz next week. ONAR 15 1. Why would a restrict digestion/enzyme help prove the Watson-Crick structure of DNA? (5 pts) S 2. Besides transformation with a plasmid as we did, how else can foreign DNA be introduced into bacteria? How is this related to the presence of bacterial restriction enzymes? (4 pts) 3. What enzymes are used in the video experiment? (2 pts) 2.5 l BamHl restriction enzyme 2.5 ul EcoRI restriction enzyme • 2.5 ul Hindill restriction enzyme 4. What is unique about the sequence a restriction enzyme recognizes? (2 pts) 5. How would cut DNA be put back together? (2 pts) 6. How does the use of restriction enzymes relate to the synthesis of the PGLO plasmid? (4 pts) 7. How are the restriction enzymes and gel kept at the same pH? (1 pt)

Answer 1

A.

1). EcoRI produces fragments of sizes 2.9, 4.5, 6.2, 7.4, and 8.0 kbp..

HindIII: Cuts twice, so you will get a small piece and a large piece.. size of 2.8kbp

PstI- 1.4bp..

2). Is already answered by appliying formula:

%w/v = w×100/ v

B.

1).Watson Crick model says that

is a double-stranded, helical molecule. It consists of two sugar-phosphate backbones on the outside, held together by hydrogen bonds between pairs of nitrogenous bases on the inside.. And To cut DNA, all restriction enzymes make two incisions, once through each sugar-phosphate backbone (i.e. each strand) of the DNA double helix.

2).

There are two ways of gene transfer by which foreign DNA can be introduced into the host cells:

1. Indirect gene transfer: When the foreign DNA is inserted into host cells by the use of a vector, the method of gene transfer is called indirect gene transfer. Indirect gene transfer generally occurs through:

(i) Plant tumours: This method has been used successfully to transform plant cells, which are difficult to transform. The gene is first inserted into the Ti plasmid of soil bacterium Agrobacterium tumefaciens. This method employs the usage of tumour causing microbe Agrobacterium tumefaciens to transfer DNA, by inducing the tumour formation in plants.

(ii) Viruses: The vector is first incorporated into a virus, which is then used to infect cells, carrying the foreign gene along with its own genetic material.

2. Direct gene transfer: The method of gene transfer in which DA is inserted directly into host cell is called direct gene transfer. Direct gene transfer occurs through:

(i) Heat shock: Cells are incubated with vector in a solution containing calcium ions at 0°C. The temperature is then suddenly raised to about 40°C. This heat shock causes some of the cells to take up the vector, as sudden heat develops pores in the membrane.

(ii) Electroporation: Cells are subjected to high-voltage pulse, which temporarily disrupts the membrane and allows the vector to enter the cell.

(iii) Gene gun: This technique fires microscopic gold or tungsten particles coated with foreign DNA at cells using a compressed air gun (burst of helium).

(Iv) Microinjection: A cell is held on a glass pipette with diameter much smaller than the cell, under a microscope and foreign DNA is injected directly into the nucleus using an incredibly fine micropipette, which punctures the plasma membrane....

Role of restriction enzyme:-

To introduce foreign DNA into a circular vector, there are three-step process:

Cutting out the gene

first remove the gene of interest from the DNA sequences on either side of it. We use restriction enzymes to do the cutting. These enzymes, which came originally from bacteria, cut DNA at specific sites in the sequence. .

Opening up the vector

By using the same restriction enzymes as they used to cut out the gene in step 1. This turns the vector into a linear molecule and makes it ready to accept the new piece of DNA.

Sticking the vector and the gene together..

3) video is not given..

4). Restriction enzymes recognises specific base sequence is known as the recognition sequence ...

Each restriction endonuclease recognises a specific
palindromic nucleotide sequences in the DNA.

For example, the following sequences reads
the same on the two strands in 5' Æ 3' direction. This is also true if
read in the 3' Æ 5' direction.
5' —— GAATTC —— 3'
3' —— CTTAAG —— 5'
Restriction enzymes cut the strand of DNA a little away from the centre of the palindrome sites, but between the same two bases on the opposite strands. This leaves single stranded portions at the ends. There are
overhanging stretches called sticky ends on each strand ..eg

EcoR1	Escherichia coli	5'GAATTC 3'CTTAAG	5'---G AATTC---3' 3'---CTTAA G---5'
BamH1	Bacillus amyloliquefaciens	5'GGATCC 3'CCTAGG	5'---G GATCC---3' 3'---CCTAG G---5'

5).When cut by the same restriction enzyme, the resultant DNA fragments have the same kind of ‘sticky-ends’ and, these can be joined together (end-to-end) using DNA ligases ...

6). Purified pGLo DNA is cut by restriction enzymes .. If we know where the enzymes will cut the DNA, ywe can predict the DNA fragment sizes and we should see on the gel, and that will help us ascertain whether we have the correct plasmid.

We can also plot Restriction maps thatthat DNA maps that show restriction sites, the locations where specific restriction enzymes will cut the DNA.

Eg:-

Created with Snap Gene Nsil (9) (2) BspDI - Clai (5307) SgrAI (5225) Afel (5105) BsmBI (5103) Pfor (5004) PfIFI - Tth1111 BsaBI (241) AfIII (301) EcoRV (386) (4748) PciI 50001 BsSHII (670) NruI (708) arac PFIMI (920) (4339) AlwNI 1000 ori AgeI (1074) BstEII (1077) BsmI (1097) araBAD promoter PGLO 5371 bp Nhel (1345) Bmti (1349) 4000 (3896) Psil NcoI - StyI (1510) MscI (1515) f1 ori (3771) DraIII GFP BsrGI (1620) 12000 Amp AmpR promoter AmpR FB Ti terminato (3423) Ahdi (3357) BsaI (3305) Bgli (3276) NmeAIII / (3248) Aset / (3200) Fspi (3054) Pvul (2942) ScaI PaeR7I - PspXI - XhoI (1765) BstBI (1966) ECORI (2063) Acc651 (2075) KpnI - TspMI - Xmal (2079) - Smal (2081) Xbal (2090) Sbfi (2106) BfUAI - BspMI (2109) SphI (2112) KfII - PpuMI (2286) 3000

7). They are kept at same pH by TBE and TAE buffer...

Two important functions performed by gel electrophoresis buffer,

1. It maintains the pH of reaction nearly neutral. The weak acid and base in buffer keep pH in the desired range.

1. By maintaining neutral pH, it controls the net charge of molecules which helps in proper migration and separation of the molecule....