Blunt end cloning
Sanjib Chaudhary
Wednesday, 19 August 2009 14:18 UTC
I am facing problems in blunt end cloning. I am hardly getting any clones.
I think ligation has not occured.For your information i am not using adapter. Please suggest.
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Replies
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First check that your ligase is working , for example check ( just on agarose gel) that the blunt ended vector ( that has not be dephosphorylated ) can be religated. If your ligase is working, then the typical problem is that your blunt ended insert contains salt or inhibitors. You can check this by adding your insert to the reaction above and establish that it does not inhibit the ligase reaction.
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vector is digested, checked in agarose gel, dephosphorylated.
Column purification is done after dephosphorylation. Vector:insert
=1:3. I don’t know if there is any salt because i do column purification wherever needed. Still then i m not getting any colonies after transformation.
pls suggest -
Hi There,
As Dawn has suggested, the only way to dissect a ligation problem like this is to use a comprehensive set of controls.
The table below, from my article on ligation troubleshooting at Bitesize Bio describes the set of controls I would recommend. For more info click here to see the original article
Best of luck,
Nick Oswald
Editor-In-Chief
Bitesize Bio – Brainfood for cell and molecular biologists
www.bitesizebio.com -
HI
FOLLOW THESE STEPS WHEN DOING BLUNT END LIGATION
1. CHECK THAT RESTRICTION ENZYME U ARE USING IS WORKING PERFECTLY. U CAN DIGEST YOUR VECTOR,WITH THE RESTRICTION ENZYME IN APPROPRIATE BUFFER.RUN ON AGROSE GEL ELECTROPHOREIS, ALSO LOAD AN UNCUT VECTOR AS A CONTROL.YOUR UNCUT VCTOR WILL GIVE 3 BANDS WHILE DIGESTED ONE SHOULD GIVE A SINGLE BAND.IF U ARE GETTING GOOD RESULT DIGEST YOUR INSERT WITH THE SAME ENZYME.ALWAYS DO TAPPING AT INTERVAL WHEN YOU ARE PERFORMING A RESTRICTION REACTION.ELUTE YOUR DESIRED VECTOR AND INSERT FROM AGROSE GEL.
2.NEXT STEP U WILL BE DOING CIP OF YOUR VECTOR. I SUGGEST U TO DO CIP OF INSERT ,NOT OF VETOR AS IT WILL LIMIT THE FORMATION OF CONCATAMERS OF YOUR INSERT. JUST IN LIAGTION REACTION U HAVE TO ADD MORE cip TRETED INSERT.2. NEXT REACTION YOU WILL BE PERFORMING IS A LIGATION REACTION. ALWAYS THAW THE LIGATION BUFFER COMPLETLY SUCH THAT NO PPT IS THERE. LIGATION BUFFER IS CONSIST OF ATP WHICH MAY BE CONSUMED ON PROLONG STORAGE.SO YOU CAN ADD EXTRA ATP.
3. AFTER TRANSFORMATION AND GROWING BACTERIAL CELL U CAN DO A SPIN AT 3000 RPM SO THAT U CONCENTRATE BATERIAL CELL IN A LOW VOLUME. AND THEN DO PLATING.
4. AFTER LIGATION YOU MAY HAVE 50 PERCENT OF CLONES IN WHICH YOUR INSERT MAY HAVE GONE IN REVERSE DIRECTION. YOU SHOULD DO COLONY PCR TAKING Fwd PRIMER COMPLEMNTRY TO VECTOR. THE PRIMER WILL BIND APPROX 300 BP UPSTREAM OF CLONING SITE. TAKE REVERSE PRIMER OF YOUR GENE.THEN DO COLONY PCR .SELECT THOSE CLONES WHICH GIVES U A PRODUCT OF 300+ YOUR GENE SIZE.HOPE IT WILL MAKE YOUR BLUNT END LIGATION MORE EASY
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HI
FOLLOW THESE STEPS WHEN DOING BLUNT END LIGATION
1. CHECK THAT RESTRICTION ENZYME U ARE USING IS WORKING PERFECTLY. U CAN DIGEST YOUR VECTOR,WITH THE RESTRICTION ENZYME IN APPROPRIATE BUFFER.RUN ON AGROSE GEL ELECTROPHOREIS, ALSO LOAD AN UNCUT VECTOR AS A CONTROL.YOUR UNCUT VCTOR WILL GIVE 3 BANDS WHILE DIGESTED ONE SHOULD GIVE A SINGLE BAND.IF U ARE GETTING GOOD RESULT DIGEST YOUR INSERT WITH THE SAME ENZYME.ALWAYS DO TAPPING AT INTERVAL WHEN YOU ARE PERFORMING A RESTRICTION REACTION.ELUTE YOUR DESIRED VECTOR AND INSERT FROM AGROSE GEL.
2.NEXT STEP U WILL BE DOING CIP OF YOUR VECTOR. I SUGGEST U TO DO CIP OF INSERT ,NOT OF VETOR AS IT WILL LIMIT THE FORMATION OF CONCATAMERS OF YOUR INSERT. JUST IN LIAGTION REACTION U HAVE TO ADD MORE cip TRETED INSERT.2. NEXT REACTION YOU WILL BE PERFORMING IS A LIGATION REACTION. ALWAYS THAW THE LIGATION BUFFER COMPLETLY SUCH THAT NO PPT IS THERE. LIGATION BUFFER IS CONSIST OF ATP WHICH MAY BE CONSUMED ON PROLONG STORAGE.SO YOU CAN ADD EXTRA ATP.
3. AFTER TRANSFORMATION AND GROWING BACTERIAL CELL U CAN DO A SPIN AT 3000 RPM SO THAT U CONCENTRATE BATERIAL CELL IN A LOW VOLUME. AND THEN DO PLATING.
4. AFTER LIGATION YOU MAY HAVE 50 PERCENT OF CLONES IN WHICH YOUR INSERT MAY HAVE GONE IN REVERSE DIRECTION. YOU SHOULD DO COLONY PCR TAKING Fwd PRIMER COMPLEMNTRY TO VECTOR. THE PRIMER WILL BIND APPROX 300 BP UPSTREAM OF CLONING SITE. TAKE REVERSE PRIMER OF YOUR GENE.THEN DO COLONY PCR .SELECT THOSE CLONES WHICH GIVES U A PRODUCT OF 300+ YOUR GENE SIZE.HOPE IT WILL MAKE YOUR BLUNT END LIGATION MORE EASY
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