I inserted 3131 bp to a plasmid with 3600 length by ECORI and BSP119I enzymes
After transformation of bacteria (TOP10) and replication, I purified the plasmid.
Plasmid digestion and sequencing showed that only 804 bp from 3131 bp was cloned.
I repeat transformation with other strain of Ecoli (BL-21) but I got the same results.
Sequencing is showed that cloning carried out at GAATTT site (804 bp) instead of GAATTC (ECORI site) at the end of 3131 bp fragment.
We were cut 3131 bp from gel after digestion with ECORI and were extracted the band from gel. therefore we removed the ECORI and not existed at ligation reaction for creation of star activity.
Can you help me?
I can think of two potential reasons;
(1) There's a DNA sequence within the 3131 bp that harms the host bacteria, whatever the mechanism is.
(2) There's a pair of repeated (or similar enough) sequences in the resulting plasmid, which directed a homologous recombination between the repeated sequences.
In both cases, cultivating the transformants at lower temperatures will help.
Also, you can try bacterial strains that are known to suppress recombinations between repeated sequences (e.g., stbl2 from Invitrogen and Sure from Agilent).
Finally, if I were you, I will design a pair of PCR primer so that I can check the presence of the insert in the plasmid by colony PCR with individual colony. That will enable you to screen a larger number of colonies at the early stage.
I can think of two potential reasons;
(1) There's a DNA sequence within the 3131 bp that harms the host bacteria, whatever the mechanism is.
(2) There's a pair of repeated (or similar enough) sequences in the resulting plasmid, which directed a homologous recombination between the repeated sequences.
In both cases, cultivating the transformants at lower temperatures will help.
Also, you can try bacterial strains that are known to suppress recombinations between repeated sequences (e.g., stbl2 from Invitrogen and Sure from Agilent).
Finally, if I were you, I will design a pair of PCR primer so that I can check the presence of the insert in the plasmid by colony PCR with individual colony. That will enable you to screen a larger number of colonies at the early stage.
Well Hiraga has nice explanation for your problem, but I have ambiguity over the sequencing accuracy for such a large fragment of DNA, Sequencing efficiency has limitation for up to 800bp, beyond that efficiency goes down. Even you will notice the error at the initial regions at 5' sequence. Tally your sequencing result with the chromatogram.
Better you check some restriction site to cleave your insert fragment to know the exact size.
Hope it will help. Good luck
The digestion of the insert or vector might be the problem.
You mentioned two restriction enzymes (EcoRI and Bsp119I) but did not mention the reaction conditions for the digestion. These two enzymes require differing reaction conditions. In the low ionic strength buffer required by Bsp119I, EcoRI will loose specificity (i.e., it will exhibit Star Activity, cutting at sites such as GAATTT.)
When performing a double digestion using Bsp119I and EcoRI, I would perform a sequential digestion, not a simultaneous digestion. First, digest for one hour with Bsp119I in its recommended reaction conditions (Buffer Tango, as recommended by Life Technologies.) Then without need for purification or inactivation, add one-tenth volume of 10X reaction buffer to the reaction mix. Then add EcoRI. Digest for one hour. After completion of digestion, purify the DNA to remove the enzymes.
Note: The Double Digestion tool on the Life Technology website offers an alternative reaction condition for double digestion by EcoRI and Bsp119I. Life Tech recommends using 2X Tango Buffer. That might work. But using those reaction conditions includes a higher risk of Star Activity than digestion in the standard reaction buffer for EcoRI. So, when using those reaction conditions, it is especially important to avoid “overdoing” the reaction. Therefore, the reaction should be of short duration (60 minutes or less), in low glycerol concentration (
sounds like you have star activity of the enzyme. Even though you band extracted the 3131bp fragment, band extraction is never 100% clean and you probably have a contaminant from the 804bp star activity cut fragment. And because this fragment is smaller, it preferentially ligates into your vector.
Repeat the digests, use less enzyme:DNA ratio. Do not leave the enzyme reacting for longer than 2 hours. When you have finished hte cloning, pick 10 colonies and run an agarose gel of the DNA before you send for sequencing. You should be able to see on a gel which is the small insert added and which is the normal plasmid added. You can always cut with BSP119I to linearise the plasmid then run the gel to clearly see the sizes. Good luck
I tend to Paul Walsh's words, the conditions you use to diges your PCR product is significant,The buffer you chosed for these two enzyme is another key contribute your cut result. you'd better to check the most suitable buffer. In the other side,check whether there are repeat sites can be cut by your enzym and design a piar of prime to detect your gene cloned into the plasmid, it is much more time and money saving than enzym.
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