I did gateway cloning and transferred my constructs into GV3101. I did several PCR confirmations of GV3101 and made glycerol stocks and stored at -80. After about 3-4 months. I am unable to amplify my genes from those stocks. Also culture grow well in 24-36 hours in liquid and 48 hours on plates but PCR appears negative.
Dear sir, There may be problem is the PCR kit, may be the primer is not working. The primers may get contaminated. You can do one thing, just Isolate plasmid and do a restriction digestion of your gene, check whether your gene is there or not.
Hello, provide more details for more reliable answers, May be problem connected also with your transformation stability.
1) Be sure that your primers work properly
2) Confirm culture identity by specific PCR for agrobacterium chromosome (16s PCR-RFLP or sequencing)
2) confirm presence of plasmid - it can be lost after cryoconservation
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