Amgen Biotech Experience Kit Instructor Checklist

Transcription

1 Rev 08_2017 Amgen Biotech Experience Kit Instructor Checklist **PLEASE FILL OUT THE DATA COLLECTION SURVEY at before beginning the sequence. Contact Information: Sherry Tsai, Greater Los Angeles Director Amgen Biotech Experience, Carol C. Fujita, Site Coordinator, CSUCI Distribution Center, All Amgen Biotech Experience labs are to be performed using all appropriate safety attire and precautions required in a science classroom laboratory. Please refer to your school district s rules regarding these. Upon PICK UP of the kit: *Check to see that you have received all the supplies listed on your supply spreadsheet ASAP. Call Carol if you are missing anything. As you unpack the boxes, note how they are packed to make it easier for you when you need to repack them (photos are included in the kit notebook). *Be sure to store supplies at the appropriate temperature. The supply spreadsheet is color coded to help you. *The kit includes a Teacher Notebook which contains all the lab updates, equipment manuals, helpful tips, and how to repack the kit. Please refer to this and the website especially for recent lab updates and teacher resources. *Please return ALL unused/left over reagents in tubes, empty bottles and supplies. Remember to keep frozen, items you received frozen. *Please keep a set of representative (or problem) agar plates in a baggie in the fridge and return with the kit. Please take photos of all Lab 4/4A gels with the camera/transilluminator set up. Do NOT erase the camera SD card. We go through the images and plates to evaluate our program. Even if you do not get good results, we use your data to figure out what went wrong. Before RETURNING the kit: *Please repack the equipment in the correct boxes. The boxes all have a list of their contents and the kit notebook has photos showing how to pack the kit. *Be sure to rinse the electrophoresis gel boxes, gel trays, gel casting board etc. with distilled water and let them AIR DRY ONLY before packing them. Remove any tape you may have put on them. DO NOT try to wipe them dry with a paper towel or ethanol! *Be sure to pack all gel combs, thermalcycler grids, SD cards, cords, gel casting board, spatula, isofreeze units, cold packs, fake ice cubes, insulated bags, empty reagent bottles, etc. as these items are sometimes forgotten. *If any pieces of equipment did not work please label them and bring it to Carol s attention. *PLEASE REFILL ALL TIP BOXES BEFORE RETURNING THE KIT. Wear gloves ONLY USED MICROFUGE TUBES, PIPET TIPS, AGAR PLATES, AND CELL SPREADERS FROM LABS 5 AND 6, THE TRANSFORMATION AND PURIFICATION LABS, ARE TO BE DISCARDED IN THE AUTOCLAVE BAGS PROVIDED. USE A TWIST TIE TO CLOSE THE BAG AND ONLY FILL EACH BAG UP TO THE WORDS AUTOCLAVE BAG. Used 1X SB buffer can be poured down the drain. All other waste (gloves, tubes, tips, etc) from labs 1,2,3 and 4 can be discarded in your regular trash. *PLEASE FILL OUT THE LAB RESULT SURVEY FORM at submit when labs are completed.

2 Amgen Biotech Experience Alternate Aliquoting Guide Lab 1 Some tools of the trade Red Practice Dye (RD) and Solutions S1, S2, S3. Distilled water. *A class set (12 tubes each) has been pre-aliquoted and can be found in one of the equipment boxes in a plastic microfuge tube box. When these solutions get low, please inform your site coordinator. ** dh 2O is used for several labs. DO NOT THROW THESE TUBES AWAY Lab 2 Preparing to clone the RFP gene: digesting the pkan-r and para plasmids 1.5 ml A para (80 ng/ul) 10uL 8uL 1.5 ml K pkan-r (80 ng/ul) 10uL 8uL 1.5 ml RE BamH I and Hind III 5uL 4uL 1.5 ml 2.5xB 2.5x restriction buffer 20uL 16uL 1.5 ml dh 2O Distilled water ** 4uL Lab 2A Preparing to verify the RFP gene: digesting the para-r plasmid 1.5 ml RP *para-r (70 ng/ul) 10uL 8uL 1.5 ml RE BamH I and Hind III 3uL 2uL 1.5 ml 2.5xB 2.5x restriction buffer 12uL 8uL 1.5 ml dh 2O Distilled water ** 2uL *There are two different tubes of para-r for the Abridged Genetic Engineering Sequence, Lab 2A uses the 70 ng/ul para-r. Check the reagent tube labels carefully. Lab 3 Building the para-r plasmid 1.5 ml 5xB 5x ligation buffer 4uL 3uL 1.5 ml LIG T4 DNA ligase 2uL 2uL 1.5 ml dh 2O Distilled water ** 2uL Lab 4 Verification of restriction and ligation using gel electrophoresis 1.5 ml M 1 kb DNA ladder/marker 8uL 8uL 1.5 ml LD 50XGelRed or 50X GelGreen loading dye 14uL 12uL Lab 4A Verification of the recombinant plasmid using gel electrophoresis 1.5 ml M 1 kb DNA ladder/marker 8uL 8uL 1.5 ml LD 50XGelRed or 50X GelGreen loading dye 8uL 6uL Feb. 2016

3 Lab 5 Transforming bacteria with the ligation products 1.5 ml LB Luria broth 350uL 300uL 1.5 ml CC *Competent cells 100uL 100uL *Do not aliquot the competent cells until class time, 15 minutes before students begin the lab. Lab 5A Transforming bacteria with recombinant plasmids 1.5 ml LB Luria broth 350uL 300uL 1.5 ml RP **para-r (10 ng/ul) 12uL 10uL 1.5 ml CC *Competent cells 100uL 100uL *Do not aliquot the competent cells until class time, 15 minutes before students begin the lab. **There are two different tubes of para-r for the Abridged Genetic Engineering Sequence, Lab 5A uses the 10 ng/ul para-r. Check the reagent tube labels carefully. Lab 5B Transforming bacteria with recombinant plasmids 1.5 ml LB Luria broth 350uL 300uL 1.5 ml RP para-r (10 ng/ul) 12uL 10uL 1.5 ml CC *Competent cells 100uL 100uL *Do not aliquot the competent cells until class time, 15 minutes before students begin the lab Lab 6 Purifying the fluorescent protein Part A 1.5 ml EC LB/amp/ara culture of E. coli *2 x 1mL 2mL 1.5 ml LyB Lysis buffer 160uL 150uL 1.5 ml EB Elution buffer 200uL 150uL *Each group will centrifuge a total of 2mL of the LB/amp/ara culture, but they will have to receive 2 aliquots of 1mL as 2mL will not fit into a 1.5mL microfuge tube. Part B *Size of tube Label tube Contents of tube *Aliquot Actually used 15mL BB Binding buffer 4M (NH 4) 2SO 4 200uL 15ml WB Wash buffer 1.3M (NH 4) 2SO 4 1mL 15mL EB Elution buffer 10mM TE 2mL 15mL CEB Column equilibration buffer 2M (NH 4) 2SO 4 2mL *You will be provided with either sets of 15mL tubes of each buffer or one large container of each buffer. If you have the 15mL tubes, one can be given to each group. If you have the larger container, divide each buffer into a set of flasks that can be shared by two groups. Colony PCR 1.5mL PCR Master mix + Taq 96uL 92uL 1.5mL + para-r 0.025ng/uL 3uL 2uL 1.5mL - para 0.025ng/uL 3uL 2uL 1.5mL M 1 kb DNA ladder/marker 9uL 8uL 1.5mL LD 50XGelRed loading dye 14uL 10uL Feb. 2016

4 AMGEN BIOTECH EXPERIENCE 20XSB Buffer Dilution and Agarose Gel Preparation ) 1X SB Buffer Preparation The Sodium Borate electrophoresis solution is sent to you at higher concentration (stock concentration) than the concentration students actually use (working concentration). To calculate the dilution from a stock concentration to a working concentration use the following method. Determine how much of the working solution you will need. Then use the formula C 1 V 1 = C 2 V 2 to determine the volume of stock solution (V 1 ) you will need to dilute to prepare the final volume of working solution (V 2 ). (C=concentration, V=volume) Example. You need to prepare 1x SB buffer (electrophoresis buffer) for 6 agarose gels. Each gel needs about 30 ml of this buffer. Amgen supplies the SB buffer, but its concentration is 20x. 30 ml x 6 gels = 180 ml Apply the formula C 1V 1 = C 2V 2 (20x)(V1) = (1x)(180mL) (V 1)= (1x) (180 ml) 20x ( V 1) = 9mL To 9.0 ml of 20x SB, add ml of dh2o (deionized or distilled) to make 180 ml of 1x SB electrophoresis buffer. 1XSB electrophoresis buffer is needed to run the gel in the electrophoresis chamber as well as to prepare the gels. Time can be saved by preparing a large volume of 1xSB buffer prior to making the gels using the formula above, or by using the table below. For Total Volume of 1X SB Use 20X SB Use Water 1000 ml 1X SB = 50 ml 20X SB 950 ml 500 ml 1X SB = 25 ml 20X SB 475 ml 200 ml 1X SB = 10 ml 20X SB 190 ml 150 ml 1X SB = 7.5 ml 20X SB ml Store the diluted buffer in several 500 ml containers to make it easy for students to pour the buffer into their electrophoresis chambers. This buffer can remain in the gel chambers and be used for several classes. Agarose Gel Preparation For each gel casting tray, you will need approximately 30 ml of a 0.8% agarose solution. Therefore, 6 gels would require 180 ml of agarose solution. To allow for some loss due to solution staying in the flask, round up to 200 ml. (Note: each MiniOne gel requires approximately 14 ml of a 0.8% agarose solution. Use this number when calculating the amount of agarose solution needed for multiple MiniOne gels.) 0.8 = x grams of agarose This is the basic formula, where? is the volume of agarose solution desired 100?mL of 1X buffer 0.8 = x grams of agarose ml of 1X buffer. (200) (0.008) = 1.6 grams of agarose (this percentage is weight of agarose over volume of 1xSB buffer) Place 1.6 grams of agarose into a 500 ml flask. Add 200 ml of 1x SB Cover the flask opening with plastic wrap. Use a pipette tip to poke a small hole in the plastic wrap. Place the covered flask in a microwave. Set the microwave for 1 minute on high. ( A hot plate can also be used to melt agarose but you will need to use a double-boiler.) As soon as boiling starts, gently swirl the flask with a gloved hand. Continue this procedure, reducing the time on the microwave (5 ± 15 seconds), until all of the agarose has been dissolved. o To check this, hold the flask to the light and swirl the solution. Look carefully for lenses of agarose crystals suspended in the liquid. If no lenses are visible, the agarose is dissolved. o If your students will pour the gels, transfer 30 ml melted agarose into 50 ml conical tubes, cap, and keep in a 60 o C water bath. Rev. 08_22_2017

5 AMGEN BIOTECH EXPERIENCE When the solution has cooled to the point that you can hold the bottom of the flask without burning your hand (60 o C), the solution can be poured into the casting trays. The solution should cover about 2 mm of the comb. Do not allow the agarose solution to cool to the point that it begins to re-solidify. If it does solidify, simply reheat the solution. o A 50 ml conical tube makes it easy to measure 30 ml for each tray o With Fotodyne electrophoresis trays or Bio-Rad trays, tape the ends of the tray with masking tape. Be sure to run your finger firmly along the masking tape to insure a tight seal. If you have OWL trays, refer to the Kit Notebook for instructions. o Combs may be placed in the gel trays prior to pouring the 60 o C agarose solution. o If the kit has a Multi-gel Casting Tray refer to those instructions. Gels will take about 30 minutes to solidify Pull the comb straight out of the gel without wiggling it back and forth. This will minimize damage to the front wall of the well. Place the gels into zip lock baggies with a small amount of 1XSB buffer. Store flat in the refrigerator until ready to use. Gels may be stored for several days (or weeks). Keep in mind that Labs 1, 4, and 4A require gels, you will need to prepare many gels to meet the needs of the labs. o o You can use a single comb per gel and have two groups load their samples in a single row. The Bio-Rad combs have fewer teeth and may require you double-comb the gel in order to get sufficient wells for two groups. If the students have not damaged (holes poked in the wells) their Lab 1 gels, they may be reused. Have the students bring the tray to you, making certain that the gel does not slide off the tray, and place the gel into a Pyrex dish or cafeteria tray in which you have 1x SB. Place all of the gels into the buffer and allow them to sit overnight. The dyes will diffuse from the gel overnight. Remove the gels and place them in zip lock baggies for Lab 4 or 4A. Store in the refrigerator until ready to use. When preparing agarose gels, you can use the formula above or use the table below. For Total Volume Use Agarose Use 1X SB 100 ml 0.8% Agarose gel 0.8 gram 100 ml 1X SB 150 ml 0.8% Agarose gel 1.2 gram 150 ml 1X SB 200 ml 0.8% Agarose gel 1.6 gram 200 ml 1X SB Problems with Warping Gel Trays Some partners have mentioned a problem with gel trays leaking agarose. In most of these cases, the problem has been traced back to pouring the agarose without first letting it cool. If the agarose is poured immediately after the agarose has melted, it is far too hot and will warp the gel tray. In some cases, we have observed hot agarose actually causing the bonds between the bottom of the tray and its sides to dissolve. The solution to this problem is to allow the agarose to cool to 60 o C or below before dispensing into the gel tray. Be certain to make your site coordinator aware of these or any other equipment problems when you return the equipment to the distribution center. Replacement gel trays cost $60. Extraneous agarose gel under the gel tray while making MiniOne gels There will be a small amount of agarose solution that leaks while casting the MiniOne gels. However, if the agarose is not allowed to cool sufficiently, the leakage will be greater. To prevent this greater leakage, allow the agarose to 60 o C or below. The small amount of agarose that solidifies under the gel tray should be wiped off prior to lowering the gel tray containing the gel into the MiniOne tank. Problems with Gel Wells When placing the gel combs in the gel tray, do not press the comb down too hard. This causes the bottom of the well to be too thin and it may break when handled. There should be about mm between the bottom of the comb and the gel tray. Rev. 08_22_2017

6 Amgen Biotech Experience Tips and Alternate Procedures Lab 1 Using the plastic pipetting practice gels and laminated practice sheets. 1. For the practice plates use water that is very slightly soapy (about 2 drops of Dawn in 500mL of water) to help prevent bubbles in the wells. Do not use 1xSB. a. Have beakers of this solution at the lab stations for groups to share. b. Gentle pressure on the wells will dislodge any bubbles that do form. 2. After use, thoroughly rinse the plates with distilled water and let air dry before returning them to the small box. Do not dry with paper towels leaves lint. 3. For the Practice Sheets, have the students place them on paper towels to prevent Red Dye being wiped onto the table top when the Practice Sheet is wiped clean. Alternate Lab 1.2 part B This alternate procedure was a component of the original version of Lab1. It allows students to practice aliquoting several solutions and mixing them in the microfuge tubes, a skill that is important in later labs. The students must also problem solve and use the results of the gel electrophoresis to determine what is in each of the original Solutions 1, 2, and 3. This alternative lab is being provided because, many teachers feel it is a valuable component of the program. Lab 5/5A/5B Proper handling of competent cells You will be receiving competent cells in an isofreeze microfuge tube container packed with cold packs in an insulated bag. The transformation efficiency of these cells is extremely high. However, it is important that these cells remain frozen until transformation day. Although you will receive the cells in the isofreeze, you'll need to keep it within the insulated bag with the cold packs, and place them all into your freezer. The insulated bag will act to buffer the changes in freezer temperatures found in frost-free freezers. We have found that schools will get excellent transformations if you are able to pick up the frozen, competent cells the day before you do transformation. Be sure to let your distribution site coordinator know if you will be picking up the competent cells when you pick up the kit or if you will be able to pick them up the day before you do the transformation lab. On the day of transformation, take out (prior to each class) only the number of tubes your class will need for transformation- each group will need 100 ul of cells; each tube contains approximately 500 ul of cells. Place these tubes in wet ice to defrost. Cells will defrost in minutes. Be certain to resuspend the cells before aliquoting. This is accomplished by gently pumping in and out with the P-200 pipette. Pre-chill the empty tubes. As the cells are aliquoted, immediately place the aliquots in wet ice. For efficient transformation, it is important the cells remain cold until the heat shock step. Lab 6 Overnight Culture alternative. If you would prefer not to grow your own Overnight Culture, indicate on your Materials Request Form that you would like your Site Coordinator to provide you with a flask of prepared culture. Your students will still do all the steps of the lab. Another alternative is asking your Site Coordinator for the prepared supernatant which allows the students to do the column chromatography, but saves some class time. Oct. 2016

7 Use of new loading dye 50XGRLD (GelRed) for Lab 4/4A: Lab 4 Student Guide page B 43 Step 6. 50XGRLD is the LD. a. Add 2.0uL of 50XGRLD to the gek-, gek+, gea-, gea+, and gelig tubes. b. Add 2.0μL of 50XGRLD to the 8μL of DNA ladder (M) to make 10uL. Load this 10μL into your gel for step 13. Teacher Guide page B 43 Aliquot Reagents for Lab 4, changes in Steps 1 and 3 Step 1. This loading dye is 50XGRLD, not the same as Solution 2. Step 3. Pipette only 8uL of DNA ladder into tubes marked M Pipette 14uL of 50XGRLD into tubes marked LD Lab 4A Student Guide page C Step 2. 50XGRLD is the LD. a. Add 2.0uL of 50XGRLD to the R- and R+ tubes. b. Add 2.0uL of 50XGRLD to the 8uL of DNA ladder (M) to make 10uL. Load this 10uL into your gel for step 7. Teacher Guide page C 27 Aliquot Reagents for Lab 4A, changes in Steps 1 and 3 Step 1. This loading dye is 50XGRLD, not the same as Solution 2. Step 3. Pipette only 8uL of DNA ladder into tubes marked M Pipette 8uL of 50XGRLD into tubes marked LD Note: Gels using 50XGRLD can be viewed on the transilluminator immediately after being run in the gel electrophoresis box. If viewing the next day, store the gels in zip baggies (without extra buffer) in the refrigerator. Gels using 50XGRLD may be disposed of in the regular trash. Rev June 2015

8 Use of new loading dye 50XGGLD (GelGreen) for Lab 4: Lab 4 Student Guide page B 43 Step 6. 50XGGLD is the LD. a. Add 2.0uL of 50XGGLD to the gek-, gek+, gea-, gea+, and gelig tubes. b. Add 2.0μL of 50XGGLD to the 8μL of DNA ladder (M) to make 10uL. Load this 10μL into your gel for step 13. Teacher Guide page B 43 Aliquot Reagents for Lab 4, changes in Steps 1 and 3 Step 1. This loading dye is 50XGGLD, not the same as Solution 2. Step 3. Pipette only 8uL of DNA ladder into tubes marked M Pipette 14uL of 50XGGLD into tubes marked LD Lab 4A Student Guide page C Step 2. 50XGGLD is the LD. a. Add 2.0uL of 50XGGLD to the R- and R+ tubes. b. Add 2.0uL of 50XGGLD to the 8uL of DNA ladder (M) to make 10uL. Load this 10uL into your gel for step 7. Teacher Guide page C 27 Aliquot Reagents for Lab 4A, changes in Steps 1 and 3 Step 1. This loading dye is 50XGGLD, not the same as Solution 2. Step 3. Pipette only 8uL of DNA ladder into tubes marked M Pipette 8uL of 50XGGLD into tubes marked LD Note: Do NOT keep the MiniOne blue light on during the entire run. Turn it on to load the gel, to check progress after 2-3 minutes, and to view the finished gel after minutes. Because the 50XGGLD is light sensitive, keeping the blue light on will cause the stain to fade and the band for the smallest fragments may not be viewable. Gels using 50XGGLD may be disposed of in the regular trash. Rev June 2015

9 Materials Return and Waste Disposal Protocol for the CSUCI ABE Site 1. Used tips and tubes from Labs 1-4 can go into your school waste. Used 1XSB may be poured down the drain. 2. Plates, tubes, tips, kimwipes and cell spreaders from Labs 5 and 6 go in the autoclave bags supplied by ABE-CSUCI. Bags should only be 2/3 full and twist tied closed. No trash. a. After emptying contents, cups/beakers used in these lab should be sprayed with 70% isopropyl alcohol solution, allowed to sit for 15 minutes, drained, washed and returned. If the teacher supplied these cups, after the 70% isopropyl alcohol treatment, they may be disposed of in the regular trash do not put them in the autoclave bags. Some plastics produce toxic fumes when autoclaved. b. Autoclave bags containing bio-waste must be returned to ABE- CSUCI for proper treatment and disposal. 3. If you did Lab 6, the flask of remaining overnight culture should be placed in the labeled zip baggie, refrigerated, and returned to ABE-CSUCI. 4. If the plasmid, enzyme, and buffer reagent tubes have anything left in them, please keep them refrigerated/frozen and return to ABE-CSUCI in the ice chest along with our isofreeze unit and ice packs. 5. Please return all other unused supplies and the 20XSB. Rev Mar_2018

10 Laboratory 1 Alternate Lab 1.2 Part B 1 Use a permanent marker to label three microfuge tubes A, B and C. 2 The table on page 1.4 summarizes the contents of each tube, but follow the directions that begin with step 3 to set up the samples. Tube dh 2 0 Solution 1 Solution 2 Solution 3 Total volume A 2 μl 4 μl 4 μl 10 μl B 2 μl 8 μl 10 μl C 2 μl 8 μl 10 μl Set the P-20 micropipette to 2 μl, add a fresh tip and dispense dh 2 O into tubes A, B and C. Eject the tip into the plastic waste container and replace with a fresh tip. Place 4 μl of Solution 1 into tube A. Eject the tip into the plastic waste container and replace with a fresh tip. Use a fresh tip and dispense 4 μl of Solution 3 into tube A. Use a fresh tip and dispense 8 μl of Solution 2 into tube B. Use a fresh tip and dispense 8 μl of Solution 3 into tube C. Save all three tubes for the next part of the lab, "Using Gel Electrophoresis to Separate Molecules".. 1.4

11 Laboratory 1 Using Gel Electrophoresis to Separate Molecules Gel electrophoresis is a method that uses an electrical current and a gel matrix (meshwork) to separate molecules such as DNA and proteins. The molecules that are being separated are either negatively charged or are made to be negatively charged. Using an electrical current, the charged molecules are then forced through a meshwork of material that will sort out the molecules according to their sizes, although molecular shape and degree of electro-negativity will influence movement through the gel. Because the molecules are negatively charged, they will migrate through the gel toward the positive (red) electrode. The more negatively charged, the faster the molecule will migrate. In this laboratory, your teacher has made a gel composed of agarose, a polysaccharide (complex sugar). The agarose is mixed with an electrolytic solution called Sodium Borate (SB). This solution contains ions, which are electrically charged atoms. These ions help conduct the electrical current through the gel. As the molecules are drawn toward the positive electrode, the smaller molecules are able to move in and around this agarose network much more quickly than the larger molecules. Thus, over the length of the gel, the molecules become separated by size Your teacher has already prepared an agarose gel for you, carefully place it into a gel tray and place the gel tray into a gel electrophoresis box. Two groups will share each gel. Take the box to the power supply you will use to run the gel. Check to make certain that the gel is positioned in the gel box so that the wells of the gel are located toward the negative (black) electrode. The dyes are negatively charged and they will move toward the positive (red) electrode. Fill the box with 1x SB buffer (there are several plastic containers containing this buffer in the lab) to a level that just covers the entire surface of the gel to a depth of 1 2 mm. Check to see that the gel is covered with buffer and that no dimples appear over the wells; add more buffer if needed. Set the micropipette to 10 μl and using a fresh tip for each sample, load each sample into a separate well. When loading each sample into the gel, center the pipette tip over the well just below the surface of the buffer. Gently depress the pipette plunger to the "first stop" to slowly expel the sample. Use your other hand to support your pipette hand to avoid shaking. Propping your elbow on the table may also help to steady your pipette hand. Because their densities are greater than the SB buffer, the dyes will sink into the wells. Draw a diagram of your gel to record which solution you placed in each well. SB Buffer Agarose gel Pipette tip Close the cover tightly over the electrophoresis chamber. Connect the electrical leads to the power supply. Be certain that both leads are connected to the same channel with the cathode ( ) to cathode (black to black) and anode (+) to anode (red to red). Turn on the power supply and set the voltage to v. After two or three minutes, look at the dyes to make certain they are moving toward the positive (red) electrode. You should begin to see the purple dye (called Bromophenol blue) beginning to separate from the blue dye (Xylene cyanole). In approximately 10 minutes, or when you can distinguish all three dyes, turn off the power switch and unplug the electrodes from the power supply. Do this by grasping the plug at the power supply not by yanking on the cord. Carefully remove the cover from the gel box so that you can better see the dyes in the gel. On you gel diagram record the banding or color pattern in each of the lanes containing your samples. Use this information to answer the "Questions". Well 1 1.5

12 Laboratory 1 Questions 1a The dyes that you separated using gel electrophoresis were: Orange G (yellow), Bromophenol blue (purple) and Xylene cyanole (blue). What electrical charge did these dyes carry? 1b What evidence allowed you to arrive at this conclusion? 2a Molecular size can play a role in separation with small molecules moving through the gel matrix more rapidly than larger molecules. The formula (or molecular) weights for these dyes are Orange G (452.38), Bromophenol blue (669.98) and Xylene cyanole (538.62). From your results, did it appear that these molecules were separated clearly on the basis of size? 2b What other factors may have played a role in the separation of these dyes? 2c Which tube contained a single dye? A, B or C? 2d Name this dye. 3 When aspirating a solution, why is it important to actually see the solution enter the pipette tip? 4a After loading your gel, did any solution remain in tubes A, B or C? 4b What could account for solution remaining in these tubes? 1.6