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Wet Lab Team

Running a Gel

• Ethidium Bromide vs. SYBR Safe Stain: Throughout the summer, we used both Ethidium Bromide and SYBR safe stain. There was no noticeable difference in the appearance of the gels and both stains seemed to produce similar results. However, it is important to note that Ethidium Bromide can be much more potent than SYBR, requiring less to be used for a single gel. If you are having issues with one in particular switch to the other to see if it will yield better results.
• Ladder: When using the ladder check to ensure that the correct amount (4 uL) is being loaded and that the ladder is diluted to the specified concentration. If you are seeing ladder that looks smudged, then the concentrated ladder may have been loaded instead of the diluted ladder.
• Gel Thickness: If you want to load the entire RD sample into the gel, try making a thicker gel. This does not mean make a gel with a higher percentage of agarose. Rather, increase the physical thickness of the gel by increasing the amount of TAE buffer and agarose. However, the percentage of the gel should still be 1%. For example, try using 120 mL of TAE buffer and 1.2 grams of agarose. This will produce thicker wells that can hold more of the RD sample.

Restriction Digest

• Increasing DNA concentration: If you are noticing that the bands on your gel are faint, try increasing the concentration of DNA in the restriction digest to 1000 nanograms. Putting more DNA into the restriction digest will allow for more of it to be cut by the restriction enzymes, hopefully giving a positive result.
• Buffers & Times: If you are noticing that you are not seeing cut plasmid on the gel, then the restriction enzymes are not cutting the DNA. There are two factors that may be causing this issue. The type of NEB Buffer you are using and the amount of time that the digests are allowed to incubate at 37 C is crucial for proper cutting. For a double digest, each enzyme combination has a specific NEB Buffer that is optimized for that particular pair of enzymes. It is important to check that you are using the correct buffer for your specific combination of enzymes. The chart below shows the enzyme combinations with the optimized buffers. The amount of time that is needed for digestion also varies for each combination of enzyme.

Ligation

• Excess Salt: Excess salt in the sample can prevent proper ligation. To remove excess salt, include an extra wash step when you do gel extraction **(5 minute wash with PE Buffer).
• False Positives: If you are getting false positives in your tranformations, try using CIP (Calf Intestinal Alkaline Phosphatase). This enzyme will remove the phosphate group from the cut backbone, preventing the backbone from re-ligating with itself. After using CIP, continue onward with ligation and transformation as normal. If colonies grow after using CIP, they have the plasmid of interest.
• Ratios & Times: If you are experiencing difficulties with ligation, try experimenting with different ratios and times. The standard ratio that we use is 3:1 (insert to backbone).
• Size of Backbone: When calculating the sixe of your backbone, be sure to include the actual part size. For example, if the backbone is 2,000 base pairs and the part itself is 120 base pairs the size of the total part is 2,120 base pairs. For the ligation calculations you would use 2,120 as the size of your backbone.

Transformation

• Gram Staining: If you notice anything odd about your overnight cultures, check with gram staining to ensure E.coli is present.
• Alpha Cells vs. TOP10 Cells: If you are having issues with your transformations try switching the cells that cells that you are using. We started off the summer using competent TOP10 cells that we had made (for ligation transformations and transformations of BioBrick parts directly from the iGEM plates). In the beginning, transformations with the TOP10 cells seemed to work with the single BioBrick parts. However, once we began to construct more complex circuits, the transformations did not seem to be working. We then switched to Alpha Cells. The Alpha cells are chemically competent and have a very high efficiency. It turns out that the TOP10 cells are fine for transforming super coiled DNA, but they do not work well for ligation transformations. We recommend, if possible, using Alpha cells for ligation transformations.

Gel Extraction

• Low Yields: If you are experiencing low yields with your gel extraction there are things that you could do to improve your results. Firstly, try to cut as close to the band of DNA as possible. This will decrease the amount of gel that the DNA is dissolved in, giving you a higher concentration of DNA per microliter. To increase yields you can also double up on the number of wells per sample. The gel slices can then be melted together and gel extraction can proceed as normal.
• QIAcube: The QIAcube is a robot that our lab purchased this summer. It can perform a variety of protocols, but we mainly use it for gel extractions and minipreps. The QIAcube was very useful when we had many samples (which was usually the case) and also helped us increase our efficiency in the lab. While the QIAcube was doing either a gel extraction or miniprep, we could continue working on other things, which was very useful. Our team is willing to be contacted for more information regarding the QIAcube.