e-gel clonewell agarose gels - thermo fisher...

Part no. 25-0967 MAN0001552

E-Gel® CloneWell™ Agarose GelsCat. nos. G6618-08, G6500ST, G6500STEU, G6500STUK

Rev. Date: 8 December 2011

E-Gel® CloneWell™ pre-cast agarose gels are designed for use with the E-Gel® iBase™ Power System, and provide a novel way to purify DNA bands. E-Gel® CloneWell™ agarose gels offer the following advantages:

• No purification necessary for downstream applications such as cloning.

• Contains the safer and environmentally friendly SYBR Safe™ DNA gel stain, so bands can be viewed using a blue-light transilluminator (such as the E-Gel® Safe Imager™ Real-Time Transilluminator, Cat. no. G6500), minimizing DNA damage.

• Precast 0.8 % agarose gels in the familiar E-Gel® format, allow fast, safe, consistent, and high-resolution separation of large and small DNA fragments.

• Eliminates the need to prepare agarose gels and buffers, and stain gels.

For additional information, or detailed operating instructions, refer to the E-Gel® Technical Guide available at www.lifetechnologies.com or contact Technical Support.

Kit ContentsEach package of E-Gel® CloneWell™ 0.8 % SYBR® Safe pre-cast agarose gels contains 18 E-Gel® CloneWell™ 0.8 % SYBR Safe® cassettes.

The E-Gel® CloneWell™ 0.8 % SYBR Safe® and iBase™ and E-Gel® Safe Imager™ Combo (G6500ST, G6500STEU, G6500STUK) contains the E-Gel® iBase™ Power System. See the E-Gel® iBase™ manual for more information.

StorageStore the gels and iBase™ at room temperature. Do not allow the temperature to drop below 4°C or rise above 40°C. Do not expose the gels unnecessarily to light.

Product UseFor research use only. Not intended for animal or human therapeutic or diagnostic use.

E-Gel® CloneWell™ Features• Two rows of wells: the top wells for loading

sample, the bottom row to retrieve your DNA.

• The reference line above the second row of wells help you time the exact moment of DNA retrieval.

• A smaller middle well for loading molecular weight marker.

General Guidelines• Load gel within 15 minutes of opening the pouch; run the gel immediately after loading.

• Use a blue-light transilluminator (such as the E-Gel® Safe Imager™ Real-Time Transilluminator) to visualize DNA.

• Monitor the band of interest carefully as it migrates near the second row of wells. It may be difficult to see low amounts of DNA in the well.

QUICK REFERENCE

250967_egel_clonewell_qrc_1Nov2011.indd 1 12/8/2011 10:17:29 AM

ProcedurePrepare Samples• Keep all sample volumes uniform and load deionized water in empty wells.

• 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™ Agarose Gel1. Plug the E-Gel® iBase™ device into an electrical outlet using the adapter plug on the base.

2. Remove the gel from the package and remove the combs.

3. Insert the gel into the base by sliding the gel into the two electrode connections on the iBase™ device. Make sure that the two electrodes on the right side of the cassette are in contact with the two contacts on the iBase™ device.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.

4. Load 20−25 µL prepared sample into well 1−8 of the top row.

5. Load 5−10 µL DNA Molecular Weight Marker into the small middle well of the top row (marked M).

6. Load 25 µL water into any remain-ing empty wells in the top row.

7. 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 DNA run time to the reference line, and run time 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 Reference Line

From Ref. Line to Collection Well

200 bp 14−18 minutes 1−2 minutes




2000 bp 21−25 minutes 1.5−2.5 minutes





Run E-Gel® CloneWell™ Agarose Gel1. 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.

2. Select the Run CloneWell program and enter the estimated Run Time to Reference Line as listed in the Run Time Table (refer to the iBase™ Power System manual for details). Press the Go button on the iBase™ device. The red light turns to a green light indicating the start of the run.

3. Monitor your gel occasionally during the run. Press the Go button to stop the run when the band of interest migrates to the printed reference line just above the bottom row of wells.

Note: If the run ends (as indicated by a flashing red light and rapid beeping) before your band has reached the reference line, press the Go button and run the gel until the band reaches the line. Press the Go button again to stop the run.

4. Once the band reaches the reference line, add more sterile water to fill the bottom row of wells (some pre-filled water is lost during the run).

5. Press the Go button, and 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 an E-Gel® Safe Imager™ Real-Time Transilluminator. At the end of this run, you may see the band of your interest migrating into the well.

Note: 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.

6. 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 into the agarose bottom. Proceed to your applica-tion using collected DNA without any further purification.

Note: If your band of interest overruns the collection well and enters the gel on the other side of the well, 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).

7. 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.

Band Size Run Time to Reference Line

From Ref. Line to Collection Well









Step 2

Step 3

Step 4

Step 5

Step 6, 7

Step 1

Imaging E-Gel® CloneWell™ Agarose Gels For photographing gels, use the SYBR Safe™ photographic filter (S37100) or SYPRO® photo graphic filter (S6656). Photograph E-Gel® CloneWell™ 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.


Purchaser NotificationLimited Use Label License: Research Use OnlyThe purchase of this product conveys to the purchaser the limited, non-transferable right to use the purchased amount of the product only to perform internal research for the sole benefit of the pur-chaser. No right to resell this product or any of its components is conveyed expressly, by implication, or by estoppel. This product is for internal research purposes only and is not for use in commercial applications of any kind, including, without limitation, quality control and commercial services such as reporting the results of purchaser’s activities for a fee or other form of consideration. For informa-tion on obtaining additional rights, please contact [email protected] or Out Licensing, Life Technologies, 5791 Van Allen Way, Carlsbad, California 92008.

DisposalSYBR Safe™ DNA gel stain shows no or very low mutagenic activity, and is not classified as hazardous waste under U.S. Federal regulations. Many institutions and have approved safe disposal of SYBR Safe™ gel stain into their waste water systems. As disposal regulations vary, contact your safety office or local municipality for appropriate SYBR Safe™ gel stain disposal in your community.

Troubleshooting

©2011 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners.

Observation Cause Solution

No current Cassette improperly inserted or is defective

Remove the gel cassette and re-insert the cassette correctly. Use 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 2- to 20-fold as described in the E-Gel® Technical Guide.

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 method (see E-Gel® Technical Guide).

Melted gel Increased current due to longer run times

Do not run the gel longer than 40 minutes.

Sample leaking from wells

Wells damaged dur-ing comb removal

Be sure to remove the comb gently without damaging the wells.

Sample overloaded Load the recommended sample volume in each well. Use the Two-Step Loading method (see the E-Gel® Technical Guide).

High background, suboptimal, or no image

No filters or wrong filter set.

See 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® with SYBR Safe™ empirically. You may need to increase the exposure time or gain setting.

Stripes vis-ible on image

No IR coating on camera lense

Use IR blocking filter or emission filter with IR coating.

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.

Headquarters5791 Van Allen Way | Carlsbad, CA 92008 USAPhone +1 760 603 7200 | Toll Free in USA 800 955 6288For support visit www.lifetechnologies.com/support

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e-gel clonewell agarose gels - thermo fisher...

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