Molecular Techniques Laboratory Lab S: Plasmid Purinieation and Re Digestion Ana

Question

Molecular Techniques Laboratory Lab S: Plasmid Purinieation and Re Digestion Analyses of Putative Cl Colony PCR of Putative Clones a. The products will be analyzed on agarose gels Part III: Restriction Digest 10X restriction enzyme buffers Plasmid DNA from Part I Sterile water and Xm Restriction enzymes (ail at 10 U/giL): Bamll, BeilI, EcoR, Ndel, Pstl, Sall, Smal, Sspl, Xbal, . Before beginning this lab, a virtual RE map of the GOI cloned in the pMAL-eSX vector sho made The instructor must approve this map before you proceed. See Figure S-2 below ce of the GOl can be analyzed using the Restriction Mapping tool available a. The DNA sequen at: http:/biotools.umassmed.edu 2. Set-up 18 restriction enzyme digests in 1.5 mL microfuge tubes for putative pMAL/GOI clones 1-8 a Select a pair of enzymes to be used for a diagnostic cut of putative clones 1-8 and the b. The sequence should be "mapped" for the presence of the RE sites listed above in Materials. and the control plasmid as shown in Table 5-2. control (tubes 10- 18). ou need to present you selections to the instructor along with your virtual restriction map. i. Y i. You will need to determine which 10X buffer is used for each enzyme or b. Mix thoroughly and then centrifuge briefly to bring contents to bottom of tube. Table 5-2 Restriction Enzyme Digests DNA Clone I Clone 2 Clone 3 Tabe Sall Ndel Ndel 1.0 1.0 10 Sal Sall Sall Clone 4 Clone 5 Clone 6 Clone 7 Clone 8 Control Clone I Clone 2 Clone 3 Clone 4 Clone 5 Clone 6 Clone 7 1.0 Ndel | 1.01 Sarl 1.0 Ndel 1.0Sall 1.0 1.0 10 12 13 1.0 1.0 15 16 17 Clone 8 Control 3. Incubate at 37°C for 1 hour. 1.0 4. Place the finished reactions at-20°C until Lab 6. a. The products will be analyzed on agarose gels.

Explanation / Answer

Calculations

Part I

Step 1: Recipe for LB + 100 microgram/ ml ampicillin glucose using straight LB and 100 mg/ml ampicillin stock solutions.

This can be calculated using the following formula

V1C1 = V2C2

Where

V1 = volume to be taken from the stock solution

C2 = Concentration of the stock solution

V2 = Final volume of the working solution

C2 = Final concentration of the working solution

Now let us prepare 50 ml of LB + 100 microgram/mL ampicillin glucose from straight LB and 100 mg/ml ampicillin

We will do it in three steps:

Step 1. Preparation of 100 microgram/ml ampicillin from 100 mg/ml ampicillin

V1C1 = V2C2

V1 = ? (X)

C1 = 100 x 10-3

V2 = 50 ml

C2 = 100 x 10-6

(X) x (100 x 10-3) = 50 x (100 x 10-6)

X = [(50 x 10-3) (100 x 10 -6)] / (100 x 10-3)

X = 50 microlitres

From the above calculation we have to take 50 microlitres of 100 mg/ml ampicillin. This will give 100 microgram/ml ampicillin concentration in LB -----------> Let us mark it as solution A (50 microlitres of 100 mg/ml ampicillin solution)

Step 2: Preparation of 100 microgram/ ml glucose

First, we will prepare 10 ml of 100 mg/ml of glucose. Weigh 1 gram of glucose and dissolve in 10 ml of distilled water. This gives 100 mg/ml concentration of glucose. Let us consider this as the stock solution.

Now we will use the earlier formula to prepare the 100 micrograms/ml of glucose:

V1C1 = V2C2

V1 = ? (X)

C1 = 100 x 10-3 glucose

V2 = 50 ml

C2 = 100 x 10-6 glucose

(X) x (100 x 10-3) = 50 x (100 x 10-6)

X = [(50 x 10-3) (100 x 10 -6)] / (100 x 10-3)

X = 50 microlitres

From the above calculation we have to take 50 microlitres of 100 mg/ml glucose. This will give 100 microgram/ml glucose concentration in LB -----------> Let us mark it as solution B (50 microlitres of 100 mg/ml glucose solution)

Step 3: Preparation of 50 ml of LB + 100 microgram/ml ampicillin glucose

Now in order to get 50 ml of LB + 100 microgram/ ml ampicillin glucose we have to do the following calculation

50 ml of LB + 100 microgram/ml ampicillin glucose (Final solution) = Solution A + Solution B + LB broth

Final solution = 50 microlitres of 100 mg/ml ampicillin + 50 microlitres of 100 mg/ml glucose + 49.9 ml of LB broth

The above underlined calculation will give 50 ml of LB + 100 microgram/ml ampicillin glucose from straight LB and 100 mg/ml stock solution.

Part 1

Step 2: The equal volume of phenol/chloroform mix

a. Volume of Solution I needed for this experiment is 2 ml (we have to add 100 microlitres to 18 tubes)

So equal volume of phenol/chloroform mix for solution I can be prepared by taking 1 ml of phenol and 1 ml of chloroform in a 2 ml centrifuge tube.

While doing the experiment for each tube we have to add 100 microlitres of phenol/chloroform mix

b. Volume of solution II needed for this experiment is 4 ml (we have to add 200 microlitres to 18 tubes)

So the equal volume of phenol/chloroform mix for solution II can be prepared by taking 2 ml of phenol and 2 ml of chloroform in a 10 ml falcon tube.

While doing the experiment for each tube we have to add 200 microlitres of phenol/chloroform mix

c. Volume of solution III needed for this experiment is 3 ml (we have to add 150 microlitres to 18 tubes)

So the equal volume of phenol/chloroform mix for solution III can be prepared by taking 1.5 ml of phenol and 1.5 ml of chloroform in a 10 ml falcon tube.

While doing the experiment, for each tube we have to add 150 microlitres of phenol/chloroform mix

Part 1

Step 19: Two volumes of ethanol based on volumes of solution I, II and III

Solution I - 100 microlitres will be used in the experiment. So, two volumes of ethanol will be 200 microlitres to each tube

Solution II - 200 microlitres will be used in the experiment. So, two volumes of ethanol will be 400 microlitres to each tube.

Solution III - 150 microlitres will be used in the experiment. So, two volumes of ethanol will be 300 microlitres to each tube.

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