實驗概要
Instructions are provided below for using the E-Gel?CloneWell pre-cast agarose gels with the E-Gel? iBase? Power System. For detailed instructions, refer to the E-Gel? Technical Guide available at www.invitrogen.com or contact Technical Support.
實驗步驟
Prepare Samples
Load 500-700 ng of total sample; load 50-200 ng DNA per band, though 20-400 ng per band is acceptable. Higher amounts require two fractions.
Load 20-25 μl total sample volume per well. Lower volumes may result in poor band shape. Excess sample may cause well to well contamination.
You may prepare DNA samples in deionized water or preferably in loading buffer; do not use a tracking dye to avoid masking the bands.
Dilute high salt samples (certain restriction enzyme and PCR buffers) 2- to 20-fold as described in the E-Gel? Technical Guide before loading.
Load E-Gel? CloneWell
Plug the E-Gel? iBase? into an electrical outlet using the adapter plug on the base.
Remove the gel from the package and insert gel (with the comb in place) into the base by sliding the gel into the two electrode connections on the iBase?. Make sure that the two electrodes on the right side of the cassette are in contact with the two contacts on the iBase?. The Invitrogen logo should be located at the bottom of the base. Press firmly at the top and bottom to seat the gel in the base. A steady, red light illuminates on the base if the gel is correctly inserted.
Pre-run the gel (with the comb in place) by selecting the program PRE-RUN 2 min and pressing the Go button on the iBase?. The red light changes to a green light indicating the start of a 2-minute pre-run. At the end of the pre-run, the current automatically shuts off.
Remove combs. Load 20-25 μl prepared sample into well 1-8 of the top row.
Load 5-10 μl DNA Molecular Weight Marker into the small middle well of the top row (marked M).
Load 25 μl water into any remaining empty wells in the top row.
Load 25-30 μl water into wells 1-8 of the bottom row, and 5-10 μl water in the middle well of the bottom row.
Estimate Run Time
Refer to the Run Time table to the right to estimate run times of your fragments to the reference line, and from the reference line to the collection well.
Note: Same bands in different wells may migrate differently; DNA fragment sizes, amounts and salt content may also slightly affect the migration rates. The run times indicated are estimates; monitor your gel occasionally during the run.
Band Size | Run Time to | From Ref. Line to |
200 bp | 14-18 minutes | 1-2 minutes |
400 bp | 15-19 minutes | 1-2 minutes |
800 bp | 17-21 minutes | 1-2 minutes |
1000 bp | 19-23 minutes | 1-2 minutes |
2000 bp | 21-25 minutes | 1.5-2.5 minutes |
3000 bp | 24-28 minutes | 1.5-2.5 minutes |
4000 bp | 28-32 minutes | 2-3 minutes |
6000 bp | 32-36 minutes | 2-3 minutes |
Run E-Gel? CloneWell
Place the E-Gel? iBase? Power System over a blue light transilluminator. Use the orange cover or orange goggles to view the bands and to avoid overexposure of your eyes to blue light.
Select the Run CloneWell program and enter the estimated Run time to Reference Line as listed in the above table (refer to the iBase? Power System manual for instructions). Press the Go button on the iBase? to run your band of interest to reach the printed reference line just above the bottom row of wells. The red light turns to a green light indicating the start of the run.
Monitor your gel occasionally during the run. If your band of interest reaches the reference line, press the Go button to stop the run. Continue with Step 5.
At the end of the run, the iBase? stops after the entered run time and displays a flashing red light and beeps rapidly. If your band did not reach the reference line, run the gel for a few more minutes until the band reaches the line.
Once the band reaches the reference line, refill the bottom row again with sterile water until the well is full (some pre-filled water is lost during the run).
Press the Go button to run the gel for the time listed in the above table until the band enters the collection well. During this period, monitor the run over a Safe Imager?. At the end of this run, you may see the band of your interest migrating into the well. We recommend monitoring the run in a darkened room for optimal results. Small DNA amounts and low molecular weight bands may be difficult to view inside the well.
Collect DNA from the well using a pipette. Be careful not to perforate the agarose bottom of the collection well. Some residual DNA will remain visible underneath the well due to migration in the garose bottom. Proceed to your application using collected DNA without any further purification. If your band of interest overruns the collection well and re-enters the gel, use the REVERSE E-Gel program of the iBase? Power System to run the band backwards into the collection well (see the iBase? Power System manual for instructions).
You may continue to collect more DNA bands from the same well (be sure to fill more water into the second row) or from other wells.
Imaging
For photographing gels, use the SYBR Safe? photographic filter (S37100) or SYPRO? photographic filter (S6656). Photograph E-Gel? CloneWell using Polaroid? 667 black-and-white print film, or image using a CCD camera or a laser-based scanner. Refer to E-Gel? Technical Guide to determine the optimal filter sets to use, or contact the instrument manufacturer for advice.
Troubleshooting
Problem | Cause | Solution |
No current | Cassette improperly inserted or is defective | Remove the gel cassette and re-insert the cassette correctly, or try using a fresh cassette. |
Poor resolution or smearing of bands | Sample overloaded | Do not load more than 200 ng of DNA per band. |
High salt samples | Dilute your samples as described in the E-Gel? Technical Guide. | |
Aberrant pre-run step | Be sure to pre-run the gel but do not exceed 2 minutes. | |
Sample not loaded properly or low sample volume loaded | Do not introduce bubbles while loading samples. For proper resolution, keep all sample volumes uniform and load water into empty wells. Use Two-Step Loading (see E-Gel? Technical Guide for details). | |
Melted gel | Increased current due to longer run times | Do not run the gel longer than 40 minutes. |
Sample leaking from wells | Wells damaged during comb removal | Be sure to remove the comb gently without damaging the wells. |
Sample is overloaded | Load the recommended sample volume per well. Use Two-Step Loading (see E-Gel? Technical Guide for details). | |
High background, suboptimal, or no image | No filters or wrong filter set | Refer to E-Gel? Technical Guide to determine the optimal filter sets to use, or contact the instrument manufacturer for advice. |
Photographic settings not optimal | Optimize settings of your system for E-Gel? CloneWell empirically. You may need to increase the exposure time or gain setting. | |
Stripes visible on image | No IR coating on camera lense. | Use IR blocking filter or emission filter with IR coating. |
Band of interest below collection well | Run time too long. | Use the REVERSE program of the iBase? Power System to run the band backwards into the collection well (see the iBase? Power System manual) |
Low volume for collection | Missed refilling water | Refill the second row with sterile water until the well is full prior to running your band of interest into the collection well. |
Low yield | Band is too big | Collect DNA from the well in two or more fractions. Be sure to load the recommended DNA amount. |
Disposal
SYBR Safe? DNA gel stain shows no or very low mutagenic activity when tested by an independent, licensed testing laboratory, and this stain is not classified as hazardous waste under U.S. Federal regulations. Many institutions and have approved safe disposal of SYBR Safe? into their waste water systems. As disposal regulations vary, contact your safety office or local municipality for appropriate SYBR Safe? disposal in your community.