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E-Gel® CloneWell Agarose Gels

2019.4.22
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18401265725

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实验概要

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.

实验步骤

Prepare Samples

Load E-Gel® CloneWell

  1. Plug the E-Gel® iBase™ into an electrical outlet using the adapter plug on the base.

  2. 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.

  3. 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.

  4. Remove combs. 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 remaining 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 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
Reference Time

From Ref. Line to
Collection Well

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.

  1. 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.

  2. 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.

  3. 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.

  4. 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).

  5. 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.

  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 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).

  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.

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.


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