BCHE341Lab
Lab 10: Isolation of bacterial plasmid, and endonuclease restriction excision of the DNA insert
1. We have prepared 3 ml overnight growths of E. coli cells containing a with an engineered extrachromosomal plasmid.
2. You will be given all the mini-prep solutions you will need to extract the plasmid from the cells. In the wheat germ protocol you extracted DNA the “old fashioned way” – i.e. without a kit. Follow the mini-prep kit directions given below EXACTLY, or you may not get enough of the plasmid for the endonuclease restriction performed in this lab.
QIAprep Spin Miniprep Kit Protocol using a microcentrifuge
This protocol is designed for purification of up to 20 ug of high-copy plasmid DNA from 1-5 ml overnight cultures of E. coli in LB (Luria-Bertani) medium.
Note: All protocol steps should be carried out at room temperature.
Procedure: You will be given 3ml of overnight growth. Centrifuge the O/N broth in a 1.5 ml eppie tube at high speed (13,000 rpm) for 5 minutes. Discard the supernant and add the remaining 1.5 ml O/N broth. Spin the tube again; bacterial cells will be pelleted out at the bottom. Discard the supernatant and proceed to following steps.
a. Resuspend pelleted bacterial cells in 250 ul Buffer P1 and transfer to a micro-centrifuge tube.
Ensure that RNase A has been added to Buffer PI. No cell clumps should be visible after resuspension of the pellet.
b. Add 250 ul Buffer P2 and gently invert the tube 4-6 times to mix.
Mix gently by inverting the tube. Do not vortex, as this will result in shearing of genomic DNA. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 min.
c. Add 350 ul Buffer N3 and invert the tube immediately but gently 4-6 times.
To avoid localized precipitation, mix the solution gently but thoroughly, immediately after addition of Buffer N3. The solution should become cloudy.
d. Centrifuge for 10 min at maximum speed in a tabletop microcentrifuge.
A compact white pellet will form.
e. Apply the supernatants from step 4 to the QIAprep column by decanting or pipetting.
f. Centrifuge for 30-60 s. Discard the flow-through.
g. Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30-60 s. Discard the flow-through.
This step is necessary to remove trace nuclease activity when using end/A*strains such as the JM series, HB101 and its derivatives, or any wild-type strain, which have high levels of nuclease activity or high carbohydrate content. Host strains such as XL-1 Blue and DH5a™ do not require this additional wash step.
h. Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30-60 s.
i. Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer.
IMPORTANT: Residual wash buffer will not be completely removed unless the flow-through is discarded before this additional centrifugation. Residual ethanol from Buffer PE may inhibit subsequent enzymatic reactions.
j. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, at first add 30 ul Buffer EB (10 mM Tris-Cl, pH 8.5) to the center of each QIAprep column, let stand for 1 min, and centrifuge for 1 min. Repeat this step with another 30 ul EB Buffer.
3. After your isolation of the plasmid DNA is complete, measure the amount of plasmid DNA you have isolated in a spectrophotometer. Make sure you record both the A260 and A280 values for your DNA solution. What does this ratio tell you?
4. Use a DNA endonuclease to cut the DNA insert out of the plasmid. The restrictions sites engineered in the plasmid are EcoRI and BamHI which you will use today to cut out the DNA insert. The restriction enzyme cuts will generates two fragments. (Plasmid map)
5. Typical Reaction set-up can be done as follows in a eppie tube-
a.15 ul ddH2O
b. 4 ul Promega Buffer E
c. 0.5 ul BSA, 100X
d. 20 ul Plasmid DNA
e. 0.5 ul Hind III
Total: 40 ul
Note- In this lab, we will provide you the master mix of all reagents except plasmid DNA.You will need to add 20 ul of your plasmid DNA into a eppie tube containing master mix.
Save the remaining DNA with proper labelling, you will use it for next lab-Gel electrophoresis.
6. When your cleavage mixture is complete, float the eppie in a 37C water bath for overnight (Digestion step). The endonucleases work best at body temperature (i.e. 37C) – why?
7. After the cleavage reaction is complete, we will freeze all of the eppie tubes. You will run the DNA gel electrophoresis of these reaction mixtures in the next lab.
8. Don’t forget to clean up before you leave!
BCHE 341L Your name here:
Lab 10: Isolation of the plasmid, and endonuclease restriction excision of the DNA insert
DNA Plasmid Isolation/Restriction Digestion Laboratory Report
1. Write down the following for your miniprep solution:
A260 reading __0.116______
A280 reading _____0.058___
A260/A280 ratio __Need to figure out and show work here_________
Also report here whether the A260/A280 ratio you reported above is a good DNA ratio. (Google what is a good DNA ratio).
Based upon your measured A260/A280 ratio what can you say about the quality of your DNA plasmid solution?
2. Look up a DNA phenolic extraction/ethanol precipitation protocol online (Googling it should bring up several lab protocols other people have put online. Describe the steps for such a protocol below, and explain for each step what is happening to the cells (what you are isolating the DNA from) and/or DNA (what you are trying to isolate).
3. What kind of nucleases are BamHI and EcoRI? Be specific.
4. What were the original sources of BamHI and EcoRI?
5. Why endonucleases work best at 37 °C?
6. Describe exactly where BamHI and EcoRI cut the DNA (i.e. the exact sequences). For this nuclease show exactly what the DNA cut ends would look like (i.e. the exact sequences). Are the ends sticky or blunt? Why?
