Recently, I have been working with some genes contained in pBlueScript II SK (+) and that contain XhoI and XbaI restriction sites. I would like to clone them in PET28a.
For instance, the digestion of both the genes and PET28a looks successful in the Agarose gel. However, after extraction I get purities below 20 ng/microliter and very low 260/280 ratios and I use these products to conduct the ligation reaction using 50 ng of digested PET28a and 37.5 ng of the gene with a total volume of 20 microliters and it is recommended to use from 2 to 5 microliters after the reaction for transformation. However, I don't observe any transforming. I have used E. coli BL21 and TOP 10.
Do you have any recommendations? Should I use higher amount of the ligation mixture?
Thanks in advance! :)
- However, after extraction
> How do you extract? May be you need to optimise the extraction protocol.
- I get purities below 20 ng/microliter and very low 260/280 ratios
> If the quality does not appear good, try to troubleshootMaybe it first before proceeding to transformation.
- and I use these products to conduct the ligation reaction using 50 ng of digested PET28a and 37.5 ng of the gene with a total volume of 20 microliters and it is recommended to use from 2 to 5 microliters after the reaction for transformation.
> What transformation system do you use? Follow the transformation protocol of the related transformation system.
> How fresh/old is your target fragment? Maybe you need to consider using fresh fragments and end repairing.
> What is your selection method for transformants? Try to use blue-white screening?
I feel like the purity of your gel extraction might be an issue here. I'd also recommend running a small amount of your product after the ligation experiment to make sure it actually worked. Do you have any positive controls for your transformation experiment? Do you use kanamycin for selection -- at what concentration?
Hi
I think that the concentration of PCR product which it clean up is low and not sufficient. I am wondering if you can do multiple PCR reaction and then combine them all into one tube and run it Gel and then do the gel purification step.
The quality of the DNA ( 260/280 ratio) is critical here. What 260/280 ratio you got? Make sure the quality of your DNA/ 260/280 ratio is okay before you proceed with your experiment further.
Run your insert gene DNA on an agarose gel and do gel extraction again.
Make sure you cut only the band of your interest.
Use extra amounts of the DNA that you want to gel extract, as there may be a big loss during the gell extraction procedure.
It is important to estimate the ratio of inset: vector. To estimate how much to use of the insert and vector run few microliters of your insert and vector on the same gel, against a DNA MW marker of known DNA concentration/ each band on the gel. Getting a good estimate of the ratio of mass of vector to insert / microliter will help you do a better ligation.
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