sequencing is alrite but still...... - (Aug/23/2006 )

Im doing transfection with normal human fibroblasts.My problem is that cells transfected with my clone r not showing any flourescence.However the control is okay.Does it mean that my clone is not good?But i've done sequencing & it shows that the sequence of the clone is alright.What should i do?

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Are you digesting your clone to have linear DNA???
Are you sure your gene or genes are in sense???

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U mean that i should tranfect DNA after digesting with one restrictin enzyme to linearize the DNA?
The sequence which i've got after sequencing is same which we were expecting.I prepared the sequence of the construct before starting cloning to ensure that everthing is in frame.So i think gene should be in sense.

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Hi,

Is your tag in frame with your insert? Where's your tag, N-term or C-term? Make sure your insert don't have a stop codon if your GTP is at the C-term (assuming the tag comes with the vector). Make sure you don't add start codon in your insert if vector has tag at N-term and its own start codon.

Don't think it's transfection problem since your control is ok.

Cheers,

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According to the sequencing results,tag is in frame with my insert.The insert is at N-terminal.My vector is a mammalian vector,how can i check expression of my protein?Can we do SDS directly after cell lysis?

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Sure. Easy method would be to "SDS boil" in Laemmli buffer, e.g. add equal vol of 2X Laemmli to cell suspension (10^6 to 10^7 cells/ml), boil for 5 - 10 min. Ice. Then spin at max speed, 4C, 10 - 15 min. Discard pellet, store s/n at -20C or use straight away.

Bests

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Thanks but can u explain it a little more coz im a new student i dnt know much.
So,i should do transfection & then cell lysis & then SDS boil.What is Laemmli buffer & how to prepare it?

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Hi,

After transfection, the next step is as follow:

For adherent: trypsinize; resuspend cell by pipetting (or swaying the flask gently); aliquot to count cell density in hemacytometer (optional if you're already gotten used to guessing the density by looking at the confluency ); spin to pellet cell; wash cell once with cold 1X PBS; add appropriate volume of 2X Laemmli buffer to cell pellet (10^6 to 10^7 cells/ml) to cell pellet; Resuspend cell in Laemmli buffer by pipetting; Boil for 5 to 10 min; Place on ice, 2 min. Spin at max speed, 10 to 15 min at 4C to pellet cell debris, aliquot supernatant to new tube and store at -20C or proceed to SDS-PAGE.

Laemmli buffer is a sample loading buffer. The recipe for it is available in the net. Working conc. is normally 1 X Laemmli.
Cheers,

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