Lab 7: Running an Agarose Gel for the Restriction Digests A) Prepare a TAE agarose gel. (A more detailed protocol is in Lab 4) 1. Prepare 80 ml of 1% agarose gel in 1X TAE Gel buffer by heating in the microwave 0.8 g of agarose in 40 ml 1X TAE and then adding another 40 ml 1X TAE to cool. 2. Once the gel has cooled to ~65 C, add 5 µl of 10 mg/ml Ethidium Bromide (EtBr) to the agarose gel mix. REMEMBER, EtBr IS A POTENTIAL MUTAGEN. ALWAYS WEAR GLOVES WHEN WORKING WITH EtBr. Continue to let the agarose slowly cool until it is not too hot to hold in your hand (approx. 60 o C). This should take about 10 minutes. During the 10-minute cooling period you should periodically swirl the bottle gently to prevent any lumps from forming due to uneven cooling. 3. Pour the cooled agarose into the gel casting box with the well combs in place and let it harden for approximately 15-20 minutes. 4. Place the hardened gel in the gel box. Fill the gel box with used 1X TAE running buffer so that the gel is submerged under approximately 0.5 inches of running buffer. Gently pull the combs out and do not discard the combs. B) Prepare samples and load gel 1. Each student should prepare uncut DNA samples for agarose gel electrophoresis. How can you tell whether the enzyme has cut or not? The best way to see this is to include on your gel a comparison lane containing uncut plasmid, i.e., plasmid DNA that has not been treated with a restriction enzyme. Prepare an uncut control sample (your original miniprep) for each plasmid clone in the following way: a) Thaw the yellow tube of miniprep DNA (tube is labeled with MP). b) Label a clear 1.7 ml microfuge tube with each clone name and a U on top to indicate that it is the uncut DNA. c) Add the following items to the U labeled tube in the order shown Sterile ddh 2 O 8 µl 10X Gel Loading Dye 2 µl Miniprep Plasmid DNA 10 µl = 20 µl ã 2017 WSSP 7-1
2. Retrieve your digested samples from the freezer (these samples were prepared in Laboratory 6). They already have 2 µl of 10X loading dye in them (Step 9, Lab 6). 3. Load your agarose gel. a. Load the uncut and digested DNA samples (all are approx. 20 µl) into adjacent wells using a P-20 pipetman set at 20 µl (see photo on bottom of this page). In addition, load one lane with 20 µl of pre-made DNA size standards known as 1Kb Plus DNA Ladder. To save DNA ladder marker and gel materials we recommend that students work together to use all the lanes on a gel. You must load your uncut sample in the lane adjacent to your cut (digested) sample so that you can easily compare the bands. b. Run the gel at 150 V. Make sure that bubbles are coming from the wire electrodes, indicating that there is current in the gel. Check the gel after a couple of minutes to make sure the dyes are running in the proper direction. c. When the dyes have migrated an appropriate distance, turn off the power supply, and remove your gel. NOTE: The running buffer can be recycled and used multiple times!!! Pour the running buffer back into the 1X TAE bottle. We often use our gel buffers 5-10 times. d. Remove Gloves! Take a photo of the gel to mock up and upload to your school s Google Docs site. Follow the steps that are described here to process the gel. i) Immediately after you take a picture of the gel, save the file, and transfer it to a computer. ii) Import the picture into PowerPoint or another graphics program and crop the image to only include the gel. iii) Import the cropped gel image into the restriction digest template slide in the RDGtemplate.ppt file. iv) Adjust the size and position to closely match the marker and Fig 1. An example of a mocked up gel. U uncut, C- lane labels. Individual PvuII The name of the clones are indicated at the top and the predicted size of the inserts are shown at the bottom. The sizes of the marker bands are also shown. ã 2017 WSSP 7-2
adjustment of the labels may be necessary. Make any changes to the text for the names of the gel, students and clones. v) Compare the uncut and digest lanes of each clone to determine if the restriction digest was successful. vi) Determine the size of each insert based on the size of the restriction digest Sue Colettavii) Save the file as a jpeg and upload a copy of the file to the appropriate box in Column G of your school s Google Docs site as described for the PCR Gel image in Lab 4 (Figure 2). BE SURE TO SET THE SETTINGS ON GOOGLE DOCS TO SHARE SO THAT EVERYONE WITH A LINK CAN SEE IT!! ix) Enter the results of the analysis of the clones into your school s Google Docs Clone Report Sheet and be sure to link the RDG.jpeg file for each clone in the report sheet. C) Interpret your restriction digest reactions A complete description about how to analyze the gel of your restriction digestions is in Chapter 2B of the lecture notes. Now that you have taken a photograph of your agarose gel, it is time to interpret the data that you obtained. The main things that you want to learn from this agarose gel electrophoresis are: 1) Was the DNA minprep of good quality? 2) Are the insert sizes found in the restriction digest experiment the same as the sizes that were predicted by the PCR? 1. To determine if the plasmid DNA miniprep was of good quality, examine the uncut lanes. There should be a strong band of supercoiled DNA. There may also be some open circle and linear bands (see Chapter 2 notes for an explanation). If the uncut band is very faint then there may not be enough DNA in the miniprep solution to get a good sequencing reaction. 2. Determine the size of the DNA fragments that result from the digest. Your digested sample should look different than your uncut Sample. If it does not, then your digest did not work. You should see at least two bands in the digest. One will be the ptriplex2 backbone fragment, which is approximately 2.9 Kb. The second band will be the cdna insert. Determine the size of the fragment by comparing it with the marker bands. Since this fragment has roughly 700 bp of vector sequences, subtract 700 from this band size to determine the size of the cdna insert. 3. Compare the size of the insert by the PCR and the RD. These should be close (<200 bp) to each other. If they are not, then you will need to recheck your analyses of both of your PCR and RD gels. ã 2017 WSSP 7-3
4. Enter your calculation of the fragment size into Column H of your school s Google Docs Clone Report sheet. If, for some reason, you can t determine the size of the insert from the digest then enter NA into the box. However, you must add a comment indicating why you could not determine the size of the insert (See Fig 2). 5. Indicate if the clone should be sent for sequencing (Yes or No) in Column I of your school s Google Docs Clone Report sheet). There must be an entry for every clone that you have performed a digest reaction with. If there are no entries in this column then the clone data will not be reviewed by the WSSP staff. Your entries into the Google-Docs sheet will first be reviewed by your teacher. If they are correct your teacher will change the box in Column I to a teal color to indicate that it is ready for review by the WSSP staff. If there is a problem with the data entry or with the analysis, the box will be colored red indicating that there is a problem. Most problems arise from links to gel images not being shared, improperly labeled gels (marker sizes or lane headings), or incorrect determination of the sizes. Fig 2. RD gel data into the Google Docs clone Report Sheet. If the size of the insert can t be determined by the digest gel, then enter in NA, but you must provide a comment for why you can t determine the size. Be sure to enter if the clone should be sequenced in column I. If everything is correct and the clone is of good quality and size to sequence, the WSSP staff will change the color of the box in column I to green to indicate that the clone is ready to be sequenced. A yellow colored box indicates that the clone is marginal in size or quality but that it can be sequenced if there is room in the sequencing tray. If they are good, then clones will be assigned to a sequencing tray, such as T1, T2, etc (Column J). A tray number designation without a date means the clone has been assigned ã 2017 WSSP 7-4
to a tray but has not been sequenced yet. Entries with a date indicate that the tray has been sequenced and the clone should be ready for analysis on DSAP. PLEASE NOTE: Sequencing trays contain 96 samples. Clones may be assigned to a tray number but will not be sequenced until we can fill the entire tray. Therefore, depending on when your school drops off your DNA and how many other samples are ready to be sequenced, your clone may not be sequenced for several weeks. If DNA samples are submitted for sequencing but the information on Google Docs about that sample is incomplete or incorrect, the clone will not be sequenced until all corrections have been made. ã 2017 WSSP 7-5