We have had clients successfully use their own lab cast gels. We have the following recommendations if not using the E-Gel system.
Cast a 2% agarose gel
A 2% agarose gel will give the appropriate separation to cleanly excise the 250-450 bp region.
Use an appropriate ladder with markers at 250 and 450 bp.
Because the protocol requires cutting the gel from 250-450 bp, using a ladder that includes these markers will give the best guides for precise cutting as well as determining when there is enough separation to stop running the gel. An example ladder is the NEB 50 bp ladder (pictured below). These are preferred over ladders that simply have 200, 300, 400, and 500 bp fragments.
Use SYBR Gold to visualize your gel
It is highly recommended to use SYBR Gold to stain your gel as this will help visualize the fragments that you intend to excise between 250-450 bp which makes the process easier. It is also very useful for troubleshooting any issues that may arise during the protocol, such as the presence of high/low molecular weight fragments or lack of on target peaks, after the targeted PCR. Compare SYBR Safe (left, middle images) with SYBR Gold (right image). SYBR Gold should be used to stain the gel after running an appropriate time as adding SYBR Gold to the gel during casting can interfere with DNA migration.
Run your gel for the appropriate amount of time with the appropriate voltage
The recommended E-Gel system can give good separation of desired and undesired products after running for 15 minutes. Lab cast gels will often require a longer running time. A general rule of thumb is 5-10V per cm distance between electrodes. This may take 1-2 hours of run time. Running the gel for too little time can make cutting the appropriate region difficult resulting in lost on target or including off target peaks. Running the gel for too much time can diffuse the on target bands making visualization difficult and increase the amount of gel that needs to be excised.
Because lab cast gels often take longer to run than the E-Gel system, if running the DNA + RNA + Protein protocol, you may run the PROTEIN LIBRARY CLEANUP I portion of the protocol during this time.