Questions about cloning techniques

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curious1
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Questions about cloning techniques

Post by curious1 » Tue May 16, 2006 4:19 pm

Hi sorry I just have some simple questions about purification/cloning techniques...

1) Are we allowed to vortex the DNA after we elute it from gel extraction? Does that shear the DNA?

2) If I wanted to run an undigested vector against a digested vector that was cut with restriction enzymes, for the undigested product would I just add water to the undigested vector or would I add all the other reagents such as the enzyme buffer, BSA except for the enzymes itself to the undigested vector reaction?

3) This question is for anyone but esp. KIM since she have read some of your responses related to this...when doing PCR what does the DNA template we are using contain? I know it is cDNA but on one of your posts you mentioned that it would be extracted from cells, how do we know we are extracting the cDNA we want? Going back to my question from my other post, so since there are multiple copies of the same gene on different chromosomes would my template DNA extracted from cells include DNA from all these chromosomes? Is the template DNA only specifically the sequence of the gene we are looking for or does it contain others? If they are pre-extracted from the lab then how can they be specific to the primers we design? Are they all purpose templates that can anneal to any primers? Sorry I am a bit confused.

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canalon
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Post by canalon » Tue May 16, 2006 4:45 pm

1/ depends! vortex is OK for plasmid, definitely not for genomic DNA
2/water is OK, or rather loading buffer :-) the rest is useless
3/Ouch, You don't really seem to have a perfect grasp of PCR. Let's start from the beginning.
Template is the DNA you add to be amplified. It can be genomic DNA, it can be cDNA (if you obtained it from a reverse transcription of some RNA), it can be the plasmid you just digesteddepends on what you put in your tube.
Primers are around 20bp long because statistically that is what you need to be sure of the specificty of their annealing site (there is a negligible chance to get a similar association of nucleotides by random only). But if you have multiple copies of the sme genes in your DNA template (multiple copy in your genome, or a template made of mixed DNA as in a microbial community) you will amplify all the genes.
Patrick

Science has proof without any certainty. Creationists have certainty without
any proof. (Ashley Montague)

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Post by curious1 » Tue May 16, 2006 5:16 pm

1)O no I think I vortexed the DNA briefly, but I measured the concentration with a nanodropper and there still seems to be something there.
2)By loading buffer do you mean the enzyme buffer that I used in my digestion reaction or the loading dye? I do add dye, but since my volume is not sufficient (my digested vector has 100ul in total after adding all the reagents), I need to add something to my undigested vector (I add 3ul) to bring up the volume like water.
3)So when you extract DNA from cells this includes a whole bunch of genes in it but only the gene you are interested will amplify because the primers we designed are specific to annealing to that sequence only right?

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Post by canalon » Tue May 16, 2006 7:54 pm

1/ Shearing doesn't destroy your DNA, it just breks it in little pieces. If you need to separate genomic DNA from plsmid,, or if you need your complete DNA for further analysis, it is a problem. But if it is simply for a PCR, it"s OK.
2/Yes loading Dye. Just add water or TE, or whatever in fact. But in the end you'll never be able to load 100ul in a well, so either you digest in a smaller volume, or you precipitate your DNA in the end, and you"ll have less to put into a well for the purification step.
3/Yes
Patrick

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Post by curious1 » Tue May 16, 2006 10:17 pm

1) So does that mean there's no chance my DNA is ok after a quick vortex? I am going to do ligation with it.
2) My digested vector reaction is 100ul but I just spread it out amongst a few wells. As for my undigested I just make a smaller volume to fit into one well.
3) I see!

Thank you so much canalon!

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Post by canalon » Wed May 17, 2006 2:28 am

1/ If your are making ligation it was probably either a PCR product or a plasmid, so no problem with that.
2/It's OK, but if you want to purify it, the more you spread it, the more agarose in the mix and the harder it gets to purify.
Patrick

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any proof. (Ashley Montague)

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Post by curious1 » Wed May 17, 2006 7:21 pm

ah, i see said the blind man...

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