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gel electrophoresis & assays help please

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gel electrophoresis & assays help please

Postby RachBio » Fri Feb 06, 2009 11:32 pm

Hi everyone!

I'm afraid i need some help again :)

i did a practical in october last year and teh report is due on monday. i've done the intro, method, apparatus but i am stuck on my results...

We were in teams of 2 and while i did the physical stuff my partner wrote everything down... however, for the last week i have not been able to contact him at all so he can explain the table of number that i have in front of me!

The practical was about alginate and how the alginate lyase gene can be broken down. we had to take the alginate lyase gene Aly from Klebsiella pneumonia in insert it into cosmid vector to produce pSP1. we used e.coli DH5alpha as teh source of pSP1.

This is a brief 8 step objectives:
1. isolate plasmid vector (pGH327) and pSP1 and purify from e.coli.
2. cut pHG327 and pSP1 with restriction endonuclease HindIII.
3. fragments of pSP1 added to cut pHG327 to allow recombination and then treated with dna ligase to reform phosphodigester linkages
4. bacterial colonies screened on MacConkey agar ti detect transformants
5. recombinants with aly gene isolated by detecting expression of alginate lyase enzyme
6. dna isolated from recombinanats, cut with retriction endonucleases adn analyse fragments on agarose gel electrophoresis (allows aly gene to be mapped on pSP1
7. PCR used to identify hindIII fragment of pSP1 with teh aly gene from bacterial extracts
8. expression of alginate lyase by recombinants will be quantified by measurement of specific enzyme activity

i am having 2 problems...

I have 2 gels from electrophoresis. I have measured how far each fragment has moved, but i know i need to do something else with this data, possibly to do with molecular weight?
Does anyone know how i can analyse this data, if i need molecular weight and if i do how i get it?

second problem is from the assays, we did a lyase assay and a protein assay. i have the levels of absorbance for each but i dot knwo what esle i need.... someone said i need to know the concentrtion of the glucose.....

for each assay we had 6 samples of 3 repeats. adn 3 sets of samples. S, C and G1 (not sure what they mean as my "partner" wont tell me...

i can get teh avergae absorbance and teh standard deviation for each sample set... but at teh moment thats about it... i ahve a table of data with an average and SD in it...

I ahve also been told i need to do a trend line on a standard curve,,b ut i'm gettting so very confused!

I dont know if i need to provide any more information, but if i do please ask and i will try my damn hardest to get it if i have it :)

any help with any of this would be great, i ahve been searching for hours without success...

if anyone wants to see the data or gel photos please ask :)

is there anyway i can repay someone for their help on here? send beer or flowers lol??

i hope to hear from someone soon. if it makes no sense please tell me and i will re-write :)

Rach x x
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Postby canalon » Sat Feb 07, 2009 1:47 pm

For your gel what you need is to calculat the size of each of your fragments. To do that you should use the measure collected from the molecular weight marker (MW) that was loaded on your gel alongside your fragments.
Then draw a graph wher on the horizontal axis you have the migration distance, and on the vertical axis the log of the size of the MW fragments. This should give you a staright line. Then just report the distance of migration of you fragments on the correct axis, and using your standard line see what size they correspond to.

Have fun.
Patrick

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any proof. (Ashley Montague)
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Re: gel electrophoresis & assays help please

Postby RachBio » Sat Feb 07, 2009 1:59 pm

Ah!!

That actually makes sense!
We used HindIII as the marker (MW) so i need to do the log of the fragments for those which we know (i found it in a text book i think - it meeans how many base pairs it is right?) on the y axis....

cm moved up the gel by each fragment on the x axis..... and plot the two against each other for the hindIII values.

Then use the cm moved by the other lanes to work out their molecular weight!

Thank you thank you thank you canaion!!

i'm sorry i kind of re-wrote what you just said but i just needed to clarify it in my head!




Also...anyone got a clue on the assays / absorption values that i have?
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Postby canalon » Sat Feb 07, 2009 8:28 pm

I think you have the MW figured out, although some points are not very correct:
HindIII is not the marker it is the enzyme that cut DNA. You can use it to cut any DNA and if you know what DNA you have, then it can be used to create a molecular weight marker. However, you understood how to use the standard, and that is what is important.

As for your assay, you do not give much information, but as far as I know what I expect for this kind of assay would be either different time points (qty of product over time) for each sample, and then the activity of your enzyme would be the slope of the time vs qty line (ie qty of product created for a given unit of time).
If there is no time involved, what you might have to do is to calculate the activity of each sample normalized by the qty of total protein. I cannot help much more without more detail about what was done.
Patrick

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Re: gel electrophoresis & assays help please

Postby RachBio » Sun Feb 08, 2009 9:26 pm

Hi Patrick!

Well, this iswhat my "partner" sent me... maybe itmakes more sense to you than me?

G123 outline 3 sets of 6 data and BSA outlines another 3 sets of 6 (there were 7 as it happens but ignore the first ones, they arnt anything) these two bits of data are put into the standard curves in exactly the same way we did the spot test.

secondly there are 3 s and 3 c on each. the 3 s are replicates using protein / enzyme isolates from the extracellular media and the 3 c's are proteins isolated from the cytosol in p53 of the handout. the 8 lanes represent each of the lBroth setups again on p53. in the correct order. it is this data u use to calculate the specific enzyme activity.

use the formulas from the trendlines of the specific curves to calculate concentrations of glucose and protein from the absorbances in c and s 1-8. get the glucose into units by dividing my its molecular mass and convert to seconds from the hour. divide this by the values derived from the protein formula to get the S.E.A. for each set up.


p53 of handout =
Set up 5 ml LBroth containing the appropriate antibiotic at the correct working concentration with and without IPTG (final concentration of 1mM). Recommend cultures and inoculums for the practical are as follows:
Inoculum Antibiotic Supplement
1 pSP1 Kan IPTG
2 pSP1 Kan
3 Red Colony Amp IPTG
4 Red Colony Amp
5 Large Clearing Zone Amp IPTG
6 Large Clearing Zone Amp
7 Small Clearing Zone Amp IPTG
8 Small Clearing Zone Amp
[2] Grow overnight at 37°C in orbital incubator.

[3] Remove 1 ml of each culture into a clean eppendorf and pellet cells by spinning in a microfuge for 2 mins. Discard the supernatant add a further 1 ml of the same sample to the tube. Repeat this process until the pellet represents 3 ml of culture.

[4] Remove the last 1ml of supernatant and place in a clean eppendorf to measure the amount of enzyme and protein excreted from the cell. Resuspend pellet in 1ml of fresh media and transfer to a clean eppendorf.

[5] Add 50l of chloroform to each resuspended pellet, mix well (using vortex) and leave at room temperature for 5 min.

[6] Pellet cell debris by centrifugation in microfuge for 10 min and full speed. Retain the supernatant to measure enzyme and protein levels in cell cytosol.


is there anything else i can provide?

i think the glucose concentrations are:
1, 0.5, 0.4, 0.3, 0.2, 0.1 but i'm not 100% sure if what i have is correct....

how can i attach teh file? it is microsoft word format...???
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Postby MrMistery » Mon Feb 09, 2009 4:04 am

rachbio,
you can attach the file in the "Word and PDF files" section of the forum (it is just above the off-topic section) and then you can link to it in this thread if you want
"As a biologist, I firmly believe that when you're dead, you're dead. Except for what you live behind in history. That's the only afterlife" - J. Craig Venter
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Postby RachBio » Mon Feb 09, 2009 4:37 am

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