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  2. → Viewing Profile: Posts: Guf

Guf

Member Since 06 Dec 2012
Offline Last Active Mar 25 2013 07:12 AM
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Posts I've Made

In Topic: How to set up simultaneous digestion?

04 February 2013 - 07:14 AM

I tried the digestion for 2 hours with this mastermix:

DNA: 1000 ng (10,9 ul)
NEBuffer 2: 2 ul
BlpI (10 000 U/ml): 1 ul
HindIII (100 000 U/ml): 1ul
H2O: 5,1 ul

and this is my result:

http://imageshack.us.../cleavagen.png/

According to NEBcutter there should be 2 fragments: 7000 bp and 90 bp... but I cant see the the 90 bp fragment :-(

What is wrong? Thank you.

In Topic: Problem with cloning - what is wrong?

09 January 2013 - 01:50 AM

According to your recommendations I have found a way of how to clone with two different RE. But I have another question, the restriction enzymes are HindIII and NotI and the distance between the places where they digest in vector is only 7 bp. Both enzymes work in different buffers so I can not digest simultaneously. First I want to digest with NotI, clean the plasmid with QIAquick PCR Purification Kit and then digest with HindIII (there will be 7 bp before HindIII restriction site so the digestion might not be problem).

​Is it necessary to perform dephosphorylation?

How to clean the vector after each digestion? Is it good idea to use QIAquick PCR Purification Kit or it is much more better to use QIAquick Gel Extraction Kit? Thank you for your answers :-)

In Topic: Problem with cloning - what is wrong?

06 January 2013 - 10:42 AM

Thank you for answers. I need to clone my insert into the HindIII place of the vector so I can not use 2 RE:|

I will try to clone to E. coli TOP10F or DH5alpha :-) Thank you for this advice.

Do you have experience with NEB CIAP which I use? How to set up reaction?

I have also doubts about the compositon of ligation mixtutes. Because of small size of insert (154 bp) and huge size of vector (7100 bp) a add 50 ng of vector DNA and only 1 ng of insert DNA and this is 3:1 ratio of vector:insert. To add such a small amouth of insert I have to dilute it and and do not believe that it is correct:| Do you have any experience with cloning such a small inserts to big vectors? Thank you, each advice is good:-)

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