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 and hb101) but I got the same results.
Sequencing is showed that cloning carried out at GAATTT site at 804 bp instead of GAATTC (ECORI site) at the end of 3131. .
pcr from ligation mixture showed that ligation 3131 to vector was correct.
I repeat transformation and grow ECOLI at low temperature (below 30˚C) but problem did not solved.
Can you help me?
My vector: PENTR11 dual selection
Insert: E1 adenovirus 5 (3131 bp)
could this be caused by star activityof the ecori due to either too much enzyme or too much glycerol in the insert preparation then positive selection of the smaller insert at the ligation stepin which case using less enzyme but cut for a longer time might help
Try internal digestion of your postive clones and check whether you getting the expected bands. If you getting the expected band then its right else you have to do ligation again. And the length of your insert also too high, so I recommend to clone in some other plasmid which length is more than 4.5kb.
Hi, I only want to point out that ypu have a very big plasmid + insert construct to transform. The BL21 or HB101 strains that you used are they suitable for transformation of large plasmids? If not it was more ease that you always clone only the small inserts that you obtained after the digestion.
When you said : "Sequencing is showed that cloning carried out at GAATTT site at 804 bp instead of GAATTC (ECORI site) at the end of 3131" I don't understand if you have another EcoRI site in the insert, is it appear after the PCR?
I think that one problem is that you find another site inside the insert so you generate a small insert, the second problem that with your cell competent strains I think that you have difficult in transforming large plasmid.
I agree with Paul, the EcoRI enzyme is known to have star activity when there is a high ratio of enzyme vs cutting site or high glycerol content in digestion mixture. In my lab, the upper limit of total volume of enzymes used in a digestion reaction is 5%. Within this limit, you can juggle between enzymes used in a reaction. And the enzyme unit to plasmid amount (microgram) is adjusted to arround 5N fold, where N refer to the number of cutting site in the plasmid.
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