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problem with cloning/restriction digest

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Re: problem with cloning/restriction digest

Postby blcr11 » Wed Mar 12, 2008 3:48 pm

I don't know if this is a good idea or not. On the presumtion that the ligation did in fact work, and that you just have a very low yield of the desired recombinant in a high background of parental vector, you could screen a large number of clones by colony screening a large number of putative clones hoping to find the one clone you need. You don't need to make minipreps to do that. You can screen directly from the plates. You probably want to make a master plate of those colonies you screen so that, in the event that you find a good clone, you can then grow it up.

My protocol for colony screening is taken from the LIC cloning manual from Novagen, which is available online. It doesn't matter that you're not using the LIC method. You should still be able to do colony screening the same way.
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Re: problem with cloning/restriction digest

Postby blcr11 » Wed Mar 12, 2008 4:50 pm

As long as I'm grasping at straws--are you buying your vector DNA directly from NEB, or are you constantly making up your own stocks and making your own preps of vector DNA? Most likely, you're buying the vector directly from NEB, but if you are--and have been for some time--making your own vector DNA preps, is it at all possible that you've accumulated a mutation at either the NdeI or XmaI site? If you can see on a gel the mcs fragment that drops out after double-digestion, then that's not the case, but the mcs fragment is kind of small and you may not be able to tell very easily. I would be very surprised if that were the case, but stranger things have happened.
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Postby canalon » Thu Mar 13, 2008 1:13 am

Just to check, because it happened to me, but how do you transform your bacteria?

Because I once did some electroporation with cuvettes that were supposed to be cleaned (but not knew), and had a surprising amount of positives in all my plates. None of them being what I was looking for...
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Re:

Postby ashlie23 » Sun Apr 06, 2008 6:04 am

Guys,

Thanks a lot for all the suggestions. Your comments are very helpful yet I am not able to figure out anything regarding getting positive clones.

blcr11--
I have been making my own pTYB2 prep, but they have been sequence confirmed and look just fine. I've been having major problems with getting positive clones. I see many multiple bands for my inserts at various sizes (such as 210, 330, 400, 250) any random number you can imagine..All less than 500. My control which is an uncut vector runs about 222 bp. Out of 16 colonies I screened, only one of them was negative (@222 bp- which is good) - but the rest of them were just anywhere and everywhere. I cant explain this behaviour. My insert is about 258bp and when inserted into the vector should be at 429 bp. My dilemma is that when I screen colonies on my control OTHER THAN uncut vector (this colony is from the digested vector with and without ligase) they run at the same length (~429) as others. IT SHOULD NOT...THERE'S NO WAY the pcr on digested vector with and without ligase would generate bands higher than MCS itself (which is 222 like I mentioned).
p.s. Also thanks for recommending the LIC protocol for colony screens from Novagen. I have not tried it yet, but will use it shortly. I've been using triton to lyse my cells.

Canalon--
I've been using GC5 supercompetent cells frm GeneChoice to transform my ligations. Following general transformation protocol

AND what's so messed up is that I have gotten positive clones with the same system, same RE sites, same primer designs, insert sizes just 4 months back.

I don't know what the heck is going on now????? Something is seriously wrong in subcloning.....

If anyone is interested I will send my gel photo with all the details..until then I just thought of sharing another bad news with you all. AT LEAST this time the background of uncut vector was significantly reduced when transforming into the cell line.

Thanks for listening y'all!!!
======================================================
blcr11 wrote:As long as I'm grasping at straws--are you buying your vector DNA directly from NEB, or are you constantly making up your own stocks and making your own preps of vector DNA? Most likely, you're buying the vector directly from NEB, but if you are--and have been for some time--making your own vector DNA preps, is it at all possible that you've accumulated a mutation at either the NdeI or XmaI site? If you can see on a gel the mcs fragment that drops out after double-digestion, then that's not the case, but the mcs fragment is kind of small and you may not be able to tell very easily. I would be very surprised if that were the case, but stranger things have happened.


canalon wrote:Just to check, because it happened to me, but how do you transform your bacteria?

Because I once did some electroporation with cuvettes that were supposed to be cleaned (but not knew), and had a surprising amount of positives in all my plates. None of them being what I was looking for...
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Re: problem with cloning/restriction digest

Postby biosci02 » Fri Apr 18, 2008 12:09 am

I just wanted to stop in and thank everybody for their help!
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Re: problem with cloning/restriction digest

Postby ashlie23 » Sun Apr 20, 2008 4:38 am

Hi guys, Just wanted to let you all know that the subcloning I was trying to do for past however many months has finally worked!! Special thanks to bclr and canalon for all the help :) Don't even ask how I got it done this time. I'm just glad it worked :))
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Re: problem with cloning/restriction digest

Postby camzie » Sat Oct 11, 2008 9:52 pm

i know im not supposed to respond but i have a similar question to ask along this discussion. i know that in general, the same restriction enzyme that is used to digest the plasmid vector is the same enzyme used to digest a dna of interest to make a cone bank. the question is BamHI is used to completely digest a plasmid while Sau3a is used to partially digest a chromosomal dna. i know that they should ligate based on their compatible sequence, but wouldn't there be a "gap" in the plasmid vector? and why would this require the use of 2 separate enzymes?
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Re: problem with cloning/restriction digest

Postby blcr11 » Sat Oct 11, 2008 10:53 pm

BamHI recognizes a 6-base sequence (GGATCC), leaving a 4-base overhang (GATC). Sau3A recognizes the 4-base sequence, GATC and leaves the same 4-base overhang that BamHI leaves; the two ends are compatible. There is no gap when a Sau3A fragment ligates into a BamHI site. You use the 4-base cutter to do the partial digest because there will be more sites and the resultant fragments will be smaller on average. Every BamHI site is also a Sau3A site, but not necessarily the other way around.
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Postby camzie » Sat Oct 11, 2008 11:56 pm

thank you for your response. however, i thought a partial digest yielded larger fragments...?
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Postby camzie » Sun Oct 12, 2008 12:12 am

oops! i know understand...thanks. i have another question ....what is the main reason for using a cDNA library rather than a genomic library to isolate a human gene from which you wish to make large quantities of the human protein in bacteria?
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Re: problem with cloning/restriction digest

Postby blcr11 » Sun Oct 12, 2008 1:38 am

If you are trying to clone part or all of an expressed gene from a eukaryote, you want to use cDNA because it is derived from mRNA. If you use genomic DNA, you may have to deal with unexpressed, intron sequences as well as the expressed parts. That's not to say you never want to use genomic DNA as a source. It depends on what you are trying to do.
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Postby camzie » Mon Oct 13, 2008 12:04 pm

Thank you kindly, i thought as much but was not quite sure.
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