Experiment 3

Restrictions Digest of Cryptochrome 1 and pMAL-c2x vector Introduction

The cryptochrome 1 gene that has been amplified by PCR must be placed into a suitable vector (carrier) then moved into a bacterial host. The bacterial host cell must be capable of propagating the vector, transcribing the gene, translating the massage into the properly folded and functional protein. These steps in the host cell are termed gene expression. In order to carry out the transfer of the gene into host cell, the vector must be cut and the newly created ends must be joined to the ends of the insert (PCR) products. This joining is accomplished by cleavage of the DNA at the specific sites which can then be re-united with each other, in this case, spliced with a DNA insert. The splicing is based on specific base pairing that positions the insert on the cuts of the vector plasmid, then ligation of the insert to the plasmid. The insertion of foreign DNA into a DNA sequences is termed recombination and this step is therefore the basis of recombination DNA technology. Cutting DNA at particular nucleotide sequences is accomplished by restriction endonucleases that recognize and cleave double strand DNA at specific sequences. The enzymes are named by their original bacterial source, fro example EcoR1 from E.coli, BamH1 from Bacillus amyloliquefaciens. This cleavage may leave blunt ends (both strands cut at the same position) or sticky ends, in which there is 5` overhang in which unpaired bases protrude on the 5`ends of cut DNA.

Examples EcorRI (from E.coli) recognize 5`.GAATTC..3` and cleaves

5`.GAATTC.3` 3`.CTTAAG.5`

Leave 5`overhang

5`.G3` 3`.CTTAA5`

3`AATTC.3` 3`G.5`

PstI (from Providencia stuartii) recognize 5`.CTGCGA..3` 5`.CTGCAG.3` 3`.GACGTC.5` Leave 5`overhang 5`.C3` 3`TGCAG.3` 3`.GACGA5` 3`C.5`

Note that in all cases the recognition sites are palindromic, meaning they read the same 5`to 3`on each strand. Put another way, they read the same forwards and reverse. They have been exploited to great advantage by genetic engineers to cut and splice inserts into vectors. If the vector is cut with same endonuclease as the insert, the sticky ends of insert complement the sticky ends of vectors. The insert can be dropped into the gab between the two sticky ends on the vector, shown in figure 3.1. The plasmid is going to be used as a vector pMAL-c2x, has been designed and built specifically for cloning and expressing inserted genes. The vector pMAL-c2X is designed to produce maltose-binding protein (MBP) fusions, where the protein of interest can be cleaved from MBP with the specific protease Factor Xa. The malE gene on this vector is deleted for the signal sequence, so the fusion protein produced remains in the cytoplasm. In this laboratory exercise you are going to double digest both PCR products (cryptochrome 1) and pMAL-c2x wit EcoR I and PstI.

Material and Methods

Materials Restriction Digest of Cryptochrome and pMAL-c2x 1. 10x buffer 2.EcoR I, 10 U/l 3.Pst I, 10 U/l 4. Nuclease free distilled water 5. PCR products (from experiment 2) 6. 2 g of pMAL-cx 7. 1.5 mL tubes 8. Pipetmen and sterile yellow tip

Method
Direct observation of PCR product formation and restriction digestion of Cry genes 1. Take 5 L of sample with 1 L of 6x loading dye perform 1% agrose gel electrophoresis for 1 h at 120 V. Note: you need to run along with molecular weight marker. 2. Then proceed for restriction digestion. 3. Take picture of the gel.

4. Take entire reaction and prepare following reaction 10 L of 10x Buffer 1.5 L EcoR I (10 U/ L stock) 1.5 L Pst I (10 U/ L stock) 45 L PCR products To 100 L d water 5. Incubate at water bath at 37 oC for two hours 6. After incubation, heat to 65 oC for 15 min 7. Store your sample at -20 oC for next experiment

Restriction Digest of Cryptochrome and pMAL-c2x Double digestion of PCR products (cryptochrome 1) This reaction should be prepared simultaneously with the vector reaction so they can be incubated at the same time. 1. Mix in a 200 L PCR tube 10 L of 10x Buffer 1.5 L EcoR I (10 U/ L stock) 1.5 L Pst I (10 U/ L stock) 50 L PCR products To 100 L d water 2. Incubate at water bath at 37 oC for two hours 3. After incubation, heat to 65 oC for 15 min 4. Store your sample at -20 oC for next experiment Double digestion of vector DNA (pMAL-c2x) 1. Mix in a 200 L PCR tube 6 L of 10x Buffer 1.5 L EcoR I (10 U/ L stock) 1.5 L Pst I (10 U/ L stock) 2 L vector DNA (pMAL-c2x) To 60 L dH2O 2. Incubate at water bath at 37 oC for two hours 3. After incubation, heat to 65 oC for 15 min. 4. Store your sample at -20 oC for next experiment