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    Hi all,

    I really need help on this. I recently did a experiment where i ran a gel electrophoresis, and then got a printed copy of the gel image, I had 7 lanes/samples but unfortunately it did not come out all well due to problems with the gel.
    lane 1 had 1kbp ladder
    lane 2 had undigested plasmid
    lane 3 had Ndel digested plasmid
    lane 4 had hindIII digested plasmid
    lane 5 had Ndel and hindIII digested plasmid
    lane 6 had PCR product
    lane 7 had 100bp ladder

    So i can only see the first lane i think and last lane/band patterns, but not all of them.

    How would I prepare standard curves and use them to calculate the sizes of uncut plasmid, single digest, double digest, and PCR product? :confused:

    please help anyone
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    (Original post by Gamewizard)
    Hi all,

    I really need help on this. I recently did a experiment where i ran a gel electrophoresis, and then got a printed copy of the gel image, I had 7 lanes/samples but unfortunately it did not come out all well due to problems with the gel.
    lane 1 had 1kbp ladder
    lane 2 had undigested plasmid
    lane 3 had Ndel digested plasmid
    lane 4 had hindIII digested plasmid
    lane 5 had Ndel and hindIII digested plasmid
    lane 6 had PCR product
    lane 7 had 100bp ladder

    So i can only see the first lane i think and last lane/band patterns, but not all of them.

    How would I prepare standard curves and use them to calculate the sizes of uncut plasmid, single digest, double digest, and PCR product? :confused:

    please help anyone
    Hey,

    You don't need standard curves for this.

    Do you have a map of your plasmid with the restriction sites on? Use that to work out how big your various fragments should be with the undigested plasmid running at the total size. Where do your primers bind on the plasmid? Use that to work out where your PCR product should be and how long it would be.

    Hope this makes sense?
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    (Original post by alleycat393)
    Hey,

    You don't need standard curves for this.

    Do you have a map of your plasmid with the restriction sites on? Use that to work out how big your various fragments should be with the undigested plasmid running at the total size. Where do your primers bind on the plasmid? Use that to work out where your PCR product should be and how long it would be.

    Hope this makes sense?
    I dont think i have a map of the plasmid, only got the gel image printed off.
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    (Original post by Gamewizard)
    I dont think i have a map of the plasmid, only got the gel image printed off.
    Erm ok...where did you get the plasmid from? Did you buy it in? If so then the map will be with the packaging it came with or you can check that out online using the produce code. If not then did you engineer the plasmid yourself? What is the vector? Where did you put in the restriction sites? If you're a student then maybe you should ask your supervisor/advisor?
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    Is this with antibody to bind to certain sites or not?
    I just ran one like that the other day, and obviously it only binds to the lanes that have the antigen it binds to.
    If not, you need something to show it up surely? If you just electrophorese then you split up the molecules by weight but won't necessarily see them?
    The ladders show that it's not a problem with the machine itself
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    (Original post by Philosoraptor)
    Is this with antibody to bind to certain sites or not?
    I just ran one like that the other day, and obviously it only binds to the lanes that have the antigen it binds to.
    If not, you need something to show it up surely? If you just electrophorese then you split up the molecules by weight but won't necessarily see them?
    The ladders show that it's not a problem with the machine itself
    This has nothing to do with antibody binding. I think you're confusing protein and DNA gels.

    Plasmids are made of DNA so this is a DNA gel. You're thinking of a protein gel which can be probed with antibodies.
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    (Original post by alleycat393)
    This has nothing to do with antibody binding. I think you're confusing protein and DNA gels.

    Plasmids are made of DNA so this is a DNA gel. You're thinking of a protein gel which can be probed with antibodies.
    You are indeed right! I wasn't thinking properly haha. I was running a protein gel to see which receptors my cancer cells are expressing. Don't remember the DNA one very well as our lab tech ran it
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    (Original post by alleycat393)
    Erm ok...where did you get the plasmid from? Did you buy it in? If so then the map will be with the packaging it came with or you can check that out online using the produce code. If not then did you engineer the plasmid yourself? What is the vector? Where did you put in the restriction sites? If you're a student then maybe you should ask your supervisor/advisor?
    Right, the plasmid pE41b (+) RFP was extracted from the e-coli cell pellet in a previous experiment. So the vector would be the bacterium e-coli then.

    I am not sure what you mean by putting in the restriction sites, but basically this is what I did.

    I prepared 4 tubes, one containing undigested plasmid, second containing plasmid digested with restriction enzyme Ndel, third containing plasmid digested with HindIII, and fourth one containing digested plasmid with both restriction enzymes (Ndel and HindIII). I also added buffer and water in them (diffrent amounts) And incubated them for 30 mins.

    Then I set up the electrophoresis apparatus and made up the agarose gel.

    Then I loaded my samples on to the gel.

    We were told to prepare standard curves and use them to calculate the sizes of the diff fragments.

    I hope it makes sense if not let me know, and I will try to ask my teacher about it.
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    (Original post by Gamewizard)
    Right, the plasmid pE41b (+) RFP was extracted from the e-coli cell pellet in a previous experiment. So the vector would be the bacterium e-coli then.

    I am not sure what you mean by putting in the restriction sites, but basically this is what I did.

    I prepared 4 tubes, one containing undigested plasmid, second containing plasmid digested with restriction enzyme Ndel, third containing plasmid digested with HindIII, and fourth one containing digested plasmid with both restriction enzymes (Ndel and HindIII). I also added buffer and water in them (diffrent amounts) And incubated them for 30 mins.

    Then I set up the electrophoresis apparatus and made up the agarose gel.

    Then I loaded my samples on to the gel.

    We were told to prepare standard curves and use them to calculate the sizes of the diff fragments.

    I hope it makes sense if not let me know, and I will try to ask my teacher about it.
    Did they mean like plot Distance travelled on gel vs. Time on a graph, then use that to work out the size of each fragment?
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    (Original post by Revd. Mike)
    Did they mean like plot Distance travelled on gel vs. Time on a graph, then use that to work out the size of each fragment?

    Yes thats what they mean, but I dont know how to do that the gel image I have is useless, because we had some problems with the gel during the practical and it came out wrong, you can only see a few bands in the first lane and then the last lane.
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    (Original post by Gamewizard)
    Yes thats what they mean, but I dont know how to do that the gel image I have is useless, because we had some problems with the gel during the practical and it came out wrong, you can only see a few bands in the first lane and then the last lane.
    Well, the preparation of the curves is very straightforward. You know the weights of each of the bands in the ladder, so measure how many mm/cm each one travelled, plot it on a graph. So for example, the 1000bp marker might have gone 1cm, 900bp 1.5 cm, 800bp 2.3 cm, 700bp 3cm (made up values), plot that on a graph of distance vs size.

    Obviously, if you have no bands in the other lanes, then you can't work out their sizes, because they're not there!
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    (Original post by Revd. Mike)
    Well, the preparation of the curves is very straightforward. You know the weights of each of the bands in the ladder, so measure how many mm/cm each one travelled, plot it on a graph. So for example, the 1000bp marker might have gone 1cm, 900bp 1.5 cm, 800bp 2.3 cm, 700bp 3cm (made up values), plot that on a graph of distance vs size.

    Obviously, if you have no bands in the other lanes, then you can't work out their sizes, because they're not there!
    Exactly this....sorry was looking at this as something being done in a research lab...
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    (Original post by Revd. Mike)
    Well, the preparation of the curves is very straightforward. You know the weights of each of the bands in the ladder, so measure how many mm/cm each one travelled, plot it on a graph. So for example, the 1000bp marker might have gone 1cm, 900bp 1.5 cm, 800bp 2.3 cm, 700bp 3cm (made up values), plot that on a graph of distance vs size.

    Obviously, if you have no bands in the other lanes, then you can't work out their sizes, because they're not there!
    Ok, so how would I know the weights of each of the bands in the ladder, do we need to work this out because i dont think its given to me, all i know is that the total amount of 1kbp ladder was approx 5 microlitre.

    and by measuring you mean by a ruler ? of each individual band on the image?

    thanks
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    (Original post by Gamewizard)
    Ok, so how would I know the weights of each of the bands in the ladder, do we need to work this out because i dont think its given to me, all i know is that the total amount of 1kbp ladder was approx 5 microlitre.

    and by measuring you mean by a ruler ? of each individual band on the image?

    thanks
    The ladder is standardized so that each band is a known weight. If it's a 1000bp ladder, you normally have the following bands: 1000bp, 900bp, 800bp, 700bp, 600bp, 500bp, 400bp, 300bp, 200bp and 100bp.

    Aye, get a rule and measure the distance travelled from the well to the band.
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    (Original post by Revd. Mike)
    The ladder is standardized so that each band is a known weight. If it's a 1000bp ladder, you normally have the following bands: 1000bp, 900bp, 800bp, 700bp, 600bp, 500bp, 400bp, 300bp, 200bp and 100bp.

    Aye, get a rule and measure the distance travelled from the well to the band.

    Thanks so much
 
 
 
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