I'm attempting to perform restriction-based cloning of my insert, using PstI and SacI to digest both the insert and the L4440 plasmid. After a double digest of the L4440 plasmid, I expected to see two bands on a 2% agarose gel: one at approximately 2700 bp and a smaller one around 88 bp. However, instead of these expected bands, I am observing a high-molecular-weight band around 20 kb near the top of the gel, along with a 350 bp fragment closer to the bottom.
To troubleshoot, I considered possible star activity, so I tried sequential digestion, reduced the incubation time, and lowered the plasmid concentration, but each attempt yielded the same band pattern.
To confirm I’m working with the correct plasmid, I designed primers specific to the L4440 sequence from the database, performed PCR, and obtained the expected band size, which suggests the plasmid identity is accurate.
Given these unexpected band patterns, what might be causing these results, and how can I address this issue?
1/ The amount of the plasmid in the prep might be low - you can still detect it by PCR but do not see it on the gel.
2/ The 20 kb band is likely chromosomal DNA that was not separated during the purification process from the plasmid fraction.
3/ The 350 bp fragment could be remnants of degraded RNA.
4/ So, if it is a high copy plasmid, repeat the purification. If it is a low copy plasmid, repeat the purification but substantialy increase the volume of cells from which you extract.
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