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Team:BU Wellesley Software/Tips
From 2011.igem.org
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<br> | <br> | ||
| - | + | <b>Tips & Tricks</b> | |
| + | |||
| + | This section is intended to help future iGEM teams with troubleshooting widely used laboratory protocols. This page addresses different situations/issues we encountered this summer and possible solutions. | ||
| + | |||
| + | Running a Gel: | ||
| + | |||
| + | <ul> | ||
| + | <li>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. | ||
| + | <li>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. | ||
| + | <li>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. | ||
| + | </ul> | ||
| + | |||
| + | Restriction Digest: | ||
| + | |||
| + | <ul> | ||
| + | <li>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. | ||
| + | <li>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. | ||
| + | |||
| + | <table border="1"> | ||
| + | <tr> | ||
| + | <td>Enzyme Combination</td> | ||
| + | <td>NEB Buffer</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>EcoRI + Xbal</td> | ||
| + | <td>NEB Buffer 4</td> | ||
| + | </tr> | ||
| + | <td>EcoRI + SpeI</td> | ||
| + | <td>NEB Buffer 2</td> | ||
| + | </tr> | ||
| + | <td>Xbal + Pstl</td> | ||
| + | <td>NEB Buffer 3</td> | ||
| + | </tr> | ||
| + | <td>SpeI + Pstl</td> | ||
| + | <td>NEB Buffer 2</td> | ||
| + | </table> | ||
| + | |||
| + | </ul> | ||
</body> | </body> | ||
</html> | </html> | ||
Revision as of 17:57, 10 September 2011
Tips & Tricks This section is intended to help future iGEM teams with troubleshooting widely used laboratory protocols. This page addresses different situations/issues we encountered this summer and possible solutions. 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.
- 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.
Enzyme Combination NEB Buffer EcoRI + Xbal NEB Buffer 4 EcoRI + SpeI NEB Buffer 2 Xbal + Pstl NEB Buffer 3 SpeI + Pstl NEB Buffer 2
