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siddharthsameer

Member Since 12 Jul 2012
Offline Last Active Nov 05 2012 12:49 AM

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Active Posts 36
Profile Views 487
Member Title Enthusiast
Age Age Unknown
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Male
Location
braunschweig, germany

About me

My research interests
molecular biotechnology especially molecular cloning

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ascacioc
06 Sep 2012 - 16:01
Janesh Gautam
03 Sep 2012 - 08:51
pito
20 Aug 2012 - 10:07
seadog
20 Aug 2012 - 10:03
Curtis
19 Aug 2012 - 20:38

Posts I've Made

In Topic: cloning into pPICZalphaA fails

26 October 2012 - 07:36 AM

HOYAJM, on 26 October 2012 - 06:38 AM, said:

bob1, on 25 October 2012 - 12:32 PM, said:

It's possible that you degraded the DNA during the isolation - it is critical that the alkaline lysis step is as short as possible (definitely less than 5 min) to prevent damage to the DNA. Digested DNA still gives signal on a spectrophotometer, but won't give you bands.

How much DNA did you run on the gel - staining method??

Did you check for the presence of an insert?

Bob has given you an excellent possibility of where you went wrong. Colonies should have yielded LOTS of plasmid DNA, but you got no DNA. Thus, your plasmid isolation was faulty.

You can explain there are other methods for screening colonies like colony PCR for an insert-specific product.

thanks but i tried all methods of plasmid isolation,but the results were the same, can you give some idea how can i write about all this.. thanks and regards

In Topic: cloning into pPICZalphaA fails

26 October 2012 - 01:24 AM

thank you so much for your reply I used 10microlitre to run on agarose but it did not show, i tried midi and mini and maxi prep using the kit also, and along with I tried other sample from my lab and i worked fine and showed the band.. Since after the transformation I had plenty of colonies and the I chose 7-8 colonies from the plate and did plasmid prep and then agarose , but no band so I cant send it for sequencing ...
since my period is over for the practical they asked me to write why did it fail to clone keratinase sequence, what could be the reasons and what could be done in the future to avoid IT...kINDLY GIVE ME SOME IDEA BECAUSE I HAVE NO EXPERIENCE IT CLONING S I HAVE TO WRITE ...i WOULD BE THANKFUL AND GREATFUL TO YOU, WAITING FOR YOUR REPLY..

In Topic: molecular cloning failure

23 October 2012 - 10:02 AM

phage434, on 23 October 2012 - 06:35 AM, said:

We can't possibly help with so little information about what you are trying to do or what you have tried.

I have sent you a personal mail..kindly reply to it. thanks

In Topic: 1:10000X dilution

12 September 2012 - 05:47 AM

calculation, on 12 September 2012 - 03:49 AM, said:

What do you mean by 1:10000 X dilution?

I have 5 uL of gel red and i need to dilute it to 1:10000X before use.

I know its a silly question but Can some one help? Also if you could tell me how to calculate then it would be very nice of you.

i think you can dilute 1:10 four times each from the previously made and then u can achieve it

In Topic: Ligation

12 September 2012 - 05:43 AM

ascacioc, on 12 September 2012 - 05:29 AM, said:

@phage: this is why I love this forum: you always find new ways others do stuff: nice beaker idea, better than my sticky tape that does not always stick, especially when you have 20 tubes Posted Image

@Sameer: while waiting for me to check all the cloning strategy from the start you can retry transformation as phage says: with SOC and good shaking, this only if you have ligation reaction left or stuff with which you can repeat the ligation reaction quickly tomorrow. Today prepare SOC media and autoclave it Posted Image

Andreea

@ andrea my supervisor asked me to wait until tommorow to see the colony, and i am mere a master student so cant say anything to her right now

...rest of the episode about my lovey supervisor tell u später

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