Question

5. You are tasked to clone the human actin gene (blue) from a cDNA clone that you obtained from a collaborator into the cloning vector termed pUC119 using the HindIII restriction enzyme. The 4 blue arrows show the HindIII recognition sequence on the Actin cDNA clone. piac CS Hindi Sphi Sofi Psti BspMI Aocl Hincll Sall Xhal BamHI Smal Xmal Acc651 Kpnl Eco531 lacz pMB1 ori M13 IG IEOORI PUC119 3162 bps CDNA Clone Amp Figure 2: a. pUC119 cloning vector; b. cDNA clone obtained from your collaborator showing Hindi II restriction sites (arrows) a. How many fragments of the cloning vector will be generated upon complete restriction digestion with HindIII? (2.5 points) b. How many fragments of the Actin gene will be generated upon complete restriction digestion of the cDNA clone with Hind III? (2.5 points)

This is what I was given to solve the question. Nothing else and I don't understand anything at all.

Answer 1

Aswer. a) There will be one fragment of cloning vector generated upon complete digestion with Hlind II find. II digestion site. x Hind II restriction as there is PMBT PUC 119 Amp Hers PUCHO tamp

puole (b) Three fragments of Actin gene will be generated upon complete restriction digestion of the cDNA clone with Hind II). Das there are 4 restriction sites CDNA to clone a c) The problem likely to be faceda is that we will not have to complete actin gene for cloning . thus it will not be expreneld" properly due to multiple restriction sites. We will get a shredded . gene which might be of no use. the strategy to clone Entre Astia 700 digestion gene & iso late fragment as the largest it will be a ajed

Date __I_I_ complete gene. aty. by reducing and enzyme time period for for this digestond we will need to prepare a serial dilution of the restriction enzyme starting with about 3-5 units of enzyme, per microgram of DNA of creating 5-10 dilutione to lit. Then set up a PCR reaction to I heat, kill the enzyme peet 10 minutes os do it manually Prepare a 0.8% agarose gel to visualize the bande & identify the complete autin band at particular dilution, Isolate the band to further pureed with cloning process. d) to select bacterial cells harboring on cloning vector - we will grow them Ton ampicillin media normal celle will die - shares bacterial while

roll- the transformed because vectos resistance gene. cells will survive contains campicillin