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In Topic: Selection of clones after ligation?
07 October 2011 - 11:32 AM
yes screen about 5-10 colonies, mini prep them then do the digestion look for the correct size then go for sequencing if they are the right clone sequence will give u the answer
In Topic: PCR problem
22 September 2011 - 08:45 AM
try the stratagene kit for pCR usually should work better
In Topic: PCR problem
22 September 2011 - 06:54 AM
try to change the kit sometimes if Dntp or the polymerase is not good then you wont get any product esp the polymerase
In Topic: Ligation problem
02 September 2011 - 03:46 AM
did u do the pcr clean up?
which gel clean up kit you are using or using exposing the gel uv for a long time? it got to be less than 20 sec ask some one to watch u while u are performing
usually qiagen is better for gel extraction when you cut the pieces purify only 2 well per column
overloading might be a problem add iso propanol in gel extraction too
which gel clean up kit you are using or using exposing the gel uv for a long time? it got to be less than 20 sec ask some one to watch u while u are performing
usually qiagen is better for gel extraction when you cut the pieces purify only 2 well per column
overloading might be a problem add iso propanol in gel extraction too
In Topic: Ligation problem
31 August 2011 - 06:31 PM
I know this it was bothering me for a mo just got it figured out. sub cloning driving me crazy all sorts of weird results.
Heres some sloution you can try
1. try the known plasmid with the same vector, just do the transforamtion
2. the cut some of the same plasmide with the enzyme you want to cut , then do the pcr clean up , do the ligation and transformation
3. cut the same plasmid and gel purify that then do the ligation and transformation
4. compare the results
I did all of those I got colonies on the uncut and cut and pcr cleaned but not on gel purified
i was using 1.5- 2% gel instead of 0.7 -0.8 % which was inhibiting the ligation also there is dna clean up kit by zymo research its called dna clean up and concentrator it works very well use that
Then I did cut the vector and run on 0.7% gel and I got positive colony
good luck
Heres some sloution you can try
1. try the known plasmid with the same vector, just do the transforamtion
2. the cut some of the same plasmide with the enzyme you want to cut , then do the pcr clean up , do the ligation and transformation
3. cut the same plasmid and gel purify that then do the ligation and transformation
4. compare the results
I did all of those I got colonies on the uncut and cut and pcr cleaned but not on gel purified
i was using 1.5- 2% gel instead of 0.7 -0.8 % which was inhibiting the ligation also there is dna clean up kit by zymo research its called dna clean up and concentrator it works very well use that
Then I did cut the vector and run on 0.7% gel and I got positive colony
good luck
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