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friendly's Content

There have been 16 items by friendly (Search limited from 09-April 19)


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#54057 Genomic Library plaques

Posted by friendly on 05 January 2010 - 10:01 PM in Genetics and Genomics

Dear All,

Using Stratagene Gigapack III plus packaging product, I have done the plating and I don't know if my plating is successfully done. Anyone has done it and have the succeeded plate photograph? Mind to share?
I am using BamH1 as the enzyme and protocol following Stockinger Lab 06/01/05.
Thank you very much.



#18379 Sequential restriction digestion of plasmid

Posted by friendly on 10 March 2009 - 09:03 AM in Molecular Cloning

any idea phage?
I have been wondering should I continue as the Kit for Library is costly ..



#17918 Sequential restriction digestion of plasmid

Posted by friendly on 05 March 2009 - 10:37 PM in Molecular Cloning

The inserts look perfectly prepared. Cloning them into a standard high copy number vector, such as pUC19 should be straightforward from here. You will get many clones, each containing thousands of bases of DNA sequence from the banana genome. You can sequence some of these. I still have not heard what the goal of doing so is. This is surely not the way, for example, to completely sequence the banana genome, but that might not be what you want to do.


Thanks Phage for the reply.
The photo I show u in lane 3 and 4 is DNA from the Dragon fruit. Apparently I didn't know what is the genome size for Dragon fruits so will u suggest me to continue? Will it be a full sequence. ? I doubt that because nobody have discover the total genomic size of it.
My goal is to construct a genomic library first before doing further action on it. Might need to look for functional gene from the library.

please reply and thanks again.



#17836 Sequential restriction digestion of plasmid

Posted by friendly on 05 March 2009 - 07:26 AM in Molecular Cloning

hihi...waiting for reply ..
please...



#17662 Sequential restriction digestion of plasmid

Posted by friendly on 04 March 2009 - 02:16 AM in Molecular Cloning

Posted Image

This is my latest gel photo taken using a digital camera.
Lane 1 : 1kb ladder
Lane 2 = lambda DNA
Lane 3 = Lambda DNA with BamH1
Lane 4 = sample A's DNA
Lane 5 = sample A's DNA with BamH1
Lane 6 = sample B
Lane 7 = sample B with BamH1

Digestion were done in 37oC for 2 hours and I ran on 1% gel with 50V.
Apparently the lambda DNA with BamH1 is not the same as the photo in fermentas website. In fermentas website, their gel photo showed that BamH1 make four cuts on lambda DNA but in my sample I see five.
What make them different?

another question is how is digestion? suitable to proceed for the next step?
Phage, is it big enough for library this time?
thank you very much.



#17495 Sequential restriction digestion of plasmid

Posted by friendly on 02 March 2009 - 10:04 PM in Molecular Cloning

Hi Phage, Thanks for your prompt reply.
Do you think I need to use other enzyme to digest the DNA ??



#17470 Sequential restriction digestion of plasmid

Posted by friendly on 02 March 2009 - 05:55 PM in Molecular Cloning

Posted Image

This is my latest gel photo....
Lane 1 = MassRuler DNA mix
lane 2 and 3 were lamda DNA and Lamda DNA with ECoRI
Lane 4 = 5ul of Banana DNA
lane 5= 5ul DNA with ECoRI
Lane 6 = 7ul of DNA
Lane 7 = 7ul with ECoRI

Any comment about this gel photo.
Let me know please..
All DNA were total genomic library.

ps- phage434, can I continue with the next step?

Thx



#17375 Sequential restriction digestion of plasmid

Posted by friendly on 01 March 2009 - 08:19 PM in Molecular Cloning

thanks phage434,
I will run another round by using 1kb ladder..hope it shows me the actual size...



#17252 Sequential restriction digestion of plasmid

Posted by friendly on 28 February 2009 - 11:03 AM in Molecular Cloning

thanks phage...
My project is basically to construct a genomic library..How would I know I would be able to get the complete coverage??
thank you ..



#17217 Sequential restriction digestion of plasmid

Posted by friendly on 27 February 2009 - 08:07 PM in Molecular Cloning

Thanks for all your reply..
meaning now I can progress to the next step??
Anyone did a genomic library before??
I also heard that it will sure get smearing when you digest Genomic DNA...any journal mentioned about this bfore?
thanks



#17129 Sequential restriction digestion of plasmid

Posted by friendly on 27 February 2009 - 04:18 AM in Molecular Cloning

Posted Image

This is my photo..
1st lane : 100bp ladder
2nd lane : Lamda DNA
3rd lane: Lamda DNA with ECoRI
4th lane : Banana DNA
5th lane : Banana DNA with ECoRI

Is this digestion normal ?

Thanks TC



#17125 Sequential restriction digestion of plasmid

Posted by friendly on 27 February 2009 - 12:58 AM in Molecular Cloning

oh..
Thanks again.
TC, later I will attach my photo which I digested with EcoRI and please advice me
I am digesting Banana DNA..
Thanks



#17103 Sequential restriction digestion of plasmid

Posted by friendly on 26 February 2009 - 06:58 PM in Molecular Cloning

Thanks for your prompt reply.
It is helpful by looking at the photo because I know that your DNA size are less than 1Kb...
I have DNA that are very big..more than 1kb at least...
have u try digest DNA that have more thab 1kb ???
Would u still see the distinct band for DNA more than 1kb??

Thanks TC

Hey

I don't think the company makes a difference as long as you stick to teh protocols. Here is a gel pic of one of the clones digested with Nde I/ Hind III. But I wonder how this would help. :D
Lane 1,2,3: digestion in triplets
4: 1 kb ladder
5: 100 bp ladder

Best
TC




#16961 Sequential restriction digestion of plasmid

Posted by friendly on 25 February 2009 - 10:49 PM in Molecular Cloning

Thanks TC for ur reply, I normally use specific buffer for the enzyme ..My enzyme is from Promega, will it be less efficient??
If you don't mind can u send me ur gel photo which contain digested DNA??
THank you very much.

Hey

We do a lot of cloning in the lab so don't really quantitate the DNA before digestions. I normally grow a 3ml culture for 12 hrs and do a miniprep without using the column and resuspend in 30ul water.

Take 0.5 ul of this DNA, 2ul NEB buffer(1X), 0.3 ul enzyme, 0.2 ul BSA(if required)(1X) and rest is water in a 20ul digest. Digest 8-13 hrs for a prep digestion and put up multiple digestions if you want more of the digested product..

I see a very distinct band on the gel.

Let me know if you want to know anything else.

Best
TC




#16837 Sequential restriction digestion of plasmid

Posted by friendly on 24 February 2009 - 10:44 PM in Molecular Cloning

Thanks TC, can u show me what is the ratio u used for restriction digest?
Normally I will put

7ul of ddH2O
1ul 10x buffer
1ul sample
1ul of Enzym (EcoRI or BamHI)

my sample concentration were around 1.8-2.0 ug/ml

will it be too little??
Please let me know your sample preparation please.
and can u see clear fragmets when digested??

Thank you very much


Hey

Maybe the DNA you are taking for setting up digestions is low in concentration, digest more of it.

Best
TC




Just curious question...
during all of your digestion...can you all see the abvious fragments on the gel ??
I couldn't view the clear fragments from the gel ...but using lamda DNA I can see the clear fragments.

I am using enzyme EcoRI and BamH1..

Thank you




#16727 Sequential restriction digestion of plasmid

Posted by friendly on 24 February 2009 - 12:40 AM in Molecular Cloning

Just curious question...
during all of your digestion...can you all see the abvious fragments on the gel ??
I couldn't view the clear fragments from the gel ...but using lamda DNA I can see the clear fragments.

I am using enzyme EcoRI and BamH1..

Thank you




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