the gel is numbered from left to right, the first clone from left is ladder, the
ID: 81482 • Letter: T
Question
the gel is numbered from left to right, the first clone from left is ladder, the second is 1RS, and so on...
4R, 5R, and 6R was one type of DNA. 7R, 8R, and 9R was with another sample of DNA.
1-why did you digest the plasmid with BamHI? What is your expectation if the plasmid is a reclosed vector without an insert? What is your expectation if your plasmid has an insert?
2-Describe your result from your gel obtained with the BamHI digest, for clone #1 and clone #2
3-why did you digest the plasmid with AlwNI? What is your expectation if the plasmid is a reclosed vector without an insert? What is your expectation if your plasmid has an insert?
4-Describe your result from your gel obtained with the AlwNI digest, for clone #1 and clone #2
5-why did you perform the double digest BamHI/AlwNI? What is your expectation if the plasmid is a reclosed vector without an insert? What is your expectation if your plasmid has an insert?
6-Describe your result from your gel obtained with the BamHI/AlwNI digest, for clone #1 and clone #2
Volume Volume Enzyme Volume Restriction sterile Plasmid DNA pBS-SKU (cuts mart) (100 ug/ml pBS-SK) BamHI pBS-SKU BamHI pBS-SK/ (cutsmart) (100 ug/ml pB 1 ul clone #2 F) ul (cut smart) clone 5R clone #1 (cutsmart) 6R clone #1 Clone 3 (cuts mart) clone I (cuts mart) 8R clone ul| (cuts mart) clone OR clone (cut smart) clone 6) After adding the water, buffer and DNAs, spin the tubes in the microcentrifuge for a second to get everything into the bottom of the tube Tip: close the caps of any tubes (2R, 5R and 8R) that do not get the BamHI enzyme so you don't accidently add it to them! 7) Add, to tube 1R, 1ul of BamHI enzyme. Mix the enzyme with the liguid at the bottom of the tube by pipetting up and down (otherwise the enzyme will sit at the bottom of the tube and will not mix with the DNA). Close the lid of the tube so you don't accidently add enzyme to it again. Discard the pipet tip 8) Continue to add BamHI enzyme to the 3R, 4R, 6R, 7R and OR tubes in the same manner as in step 7 and down to mix and using a new pipet tip to pipet for each enzyme addition so you do not contaminate the enzyme stock. Tip: Close the caps of tubes 1R, 4R and 7R, open the 2R, 5R and 8R tubes 9) Add the AlwNI enzyme to the 2R, 3R, 5R, 6R, 8R and OR tubes in the same manner as in step 7 (104)Explanation / Answer
1) and 3) so that we can check whether and how many Bam H1 site is present in vector. if our plasmid has an insert then after single digestion either with Bam H1 or AlwN1 we will get upshift in DNA band as compare to vector control digested with the same restriction enzyme.
2) for clone no 1 digested with either with Bam H1 or AlwN1 or with both the enzymes, we got the same band size as compare to vector control digested with the same restriction enzyme. means.it has no insert.
3) for clone no 2 digested with either with Bam H1 or AlwN1, we got 2 bands, suggesting that our insert also contains restriction sites for both the enzymes resulting two visible bands in the gel.
4) for clone no 2 digested with Bam H1 and AlwN1, we got 3 bands, suggesting that our insert also contains restriction sites for both the enzymes resulting three visible bands in the gel.
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