Recently, I have performed the LR clonase reaction using pDONR221-based vector and pET-DEST-53. No colony was grown after reaction. What is the possible cause for this situation since I don't have pENTR-gus as a positive control, thus, I'm not sure whether the problem came from pDONR or pET-DEST. Thank you guys.
Three things I will suggest to check.
1. Check the efiificiency of the competent cell. This is one of the major problem for gateway cloning. It is better to prepare fresh competent cell.
2. The LR clonase should be used very carefully. it is better to keep aliquoted LR clonase in -20 degree and use just before setiing the reaction.
3. The concentration of both entry and destination vector should be 150 ng in the 10 ul reaction.
you should not face problem to get the positive clone if you follow these things.
Hi,
Use pET-DEST with another entry clone. This will show you if the destination plasmid causes the trouble. At least, you should check the att sites in both plasmids by sequencing. This would be an alternative if you have no other entry clones.
Do you have any other destination plasmids? You could use them in the LR reaction to show that your entry clone is fine. However, I would expect that the entry clone is erroneous, because you had problems during the cloning.
The transformation efficiency could be check by using another plasmid e.g. the entry clone. However, it would be better to use a plasmid with the same antibiotic resistance to exclude issues related to the selection media.
Good luck!
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