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  2. → Viewing Profile: Posts: VJbiofication

VJbiofication

Member Since 07 Feb 2012
Offline Last Active Nov 27 2012 02:54 AM
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Posts I've Made

In Topic: Transfer buffer as running buffer?

27 November 2012 - 02:52 AM

View PostVJbiofication, on 09 November 2012 - 06:46 AM, said:

I did the same mistake today and I've run the gel with Transfer buffer instead of Running buffer. We don't use any SDS in the transfer buffer. But the marker bands and the Sample buffer was run perfectly as usual without any problem(by observation of the gel). And I am transferring now with transfer buffer to PVDF. I don't know how the blot is gonna look like.
Any suggestions or comments?

luckily the gel was run fine and even the western blot turned out to be good. So I guess using transfer buffer for running is not such a big mistake.

In Topic: Transfer buffer as running buffer?

09 November 2012 - 06:46 AM

I did the same mistake today and I've run the gel with Transfer buffer instead of Running buffer. We don't use any SDS in the transfer buffer. But the marker bands and the Sample buffer was run perfectly as usual without any problem(by observation of the gel). And I am transferring now with transfer buffer to PVDF. I don't know how the blot is gonna look like.
Any suggestions or comments?

In Topic: Transfer of proteins from gel to PVDF for short period

28 June 2012 - 12:39 AM

Thank you for your answer, but my question was about the optimal time for transfer of some amount of proteins to PVDF and leaving the remaining on gel. I can't do in-gel western(I need to take spots for MS analyses, will interfere) and I know that transfer depends on size of proteins.

In Topic: Sequential restriction digestion of plasmid

22 June 2012 - 07:12 AM

View Postphage434, on 22 June 2012 - 03:51 AM, said:

You can clone with the same RE site on both ends of your construct, but it has several disadvantages.  First, you have no control over the orientation of the insert -- it can go in forwards or backwards.  Second, since you cut the vector only once, the vector can easily (and favorably!) religate rather than ligating your insert.  This can sometimes be controlled by phosphate treatment of the vector after cutting, but this is (in my opinion) inferior in every way to using distinct enzymes at each end of your insert.  The two enzymes must leave incompatible overhangs (that is, they cannot be isoschizimers).  I'd recommend choosing enzymes that can be heat killed, avoiding a cleanup after RE digestion.  The two RE digestions can be performed simultaneously in the same reaction in most cases (and you should choose enzymes that allow this).  Most NEB enzymes now cut in buffer 4, for example.

That is a very good explanation. I don't know why, but I didn't come across this kind of information in standard materials. Thank you.

I have another question: So as you said we have to have 2 different restriction sites on both flanks of the insert. So when I prepare/order the cDNA should I also include these sequences in the final insert:  restriction site-start codon-insert-stop codon-restriction site??  Or how is it done? Please explain.

In Topic: Sequential restriction digestion of plasmid

22 June 2012 - 02:13 AM

Hello people,

I just started to learn cloning. As I know, we incorporate same restriction sites at both ends of cDNA and digest with the one enzyme.
Can you tell me why do we need to do double digestion of cDNA? How to add 2 restriction sites in the same cDNA?
What is the use/application?

and the next question is: If we use double digestion in cDNA, how the plasmid will be manipulated? I mean, do we have to do the same double digestion with plasmids? Because we need to add both plasmids and cDNA to have the same cohesive ends. How/where a double digested cDNA will be in the final plasmid vector?

My questions may seem amateurish, but please help me with my questions. Or at least give me some links where I can read about this.

Thanks.
VJ.

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