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Cloning issue


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#1 jcass5080

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Posted 01 March 2011 - 08:38 AM

I have a 3.4Kb PCR frag with HindIII at the ends. I have digested both the vector and PCR frag with HindIII (2hrs at 37C). SAP treated the vector (1hr at 37C). Ligated at RT for 2hrs. After transformation and overnight at 37C the vector only plate has quite a few colonies which I understand indicates either poor digestion/SAP treatment. But, the number of colonies decreases on the vector+insert plates (100 colonies on vector only vs. 20 colonies on vector+insert plate).

Does anyone know what might be causing this decrease?

#2 perneseblue

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Posted 01 March 2011 - 08:29 PM

Was the same amount of vector DNA used in both transformations?
Different amount of vector in the ligation mix, results in different number of colonies.

Was the PCR fragment purified before being added to the ligation mix?
PCR buffer does contain detergent which does kill bacteria cell if carried over into the ligation mix.

Was the vector gel purified?
1hr in SAP is rather long. If the vector is not gel purified uncut DNA can get into the ligation mix. Denature plasmid DNA is resistant to digest but will transform just fine.
May your PCR products be long, your protocols short and your boss on holiday

#3 Qundo12

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Posted 07 March 2011 - 12:41 AM

Beside what perneseblue mentioned, you should consider this table: http://www.neb.com/n...ucleotides.asp. It's about the sequences flanking the ends of the oligonucleotides that are necessary for the digestion. In some cases the linear DNA needs specific extra nucleotides for the efficient digestion. With HindIII, it needs CCC if HindIII site is on the 5' end or GGG if HindIII site is on the 3' end, otherwise the result is 0% digested DNA even after 20 hours of incubation.
If you didn't notice that, the best way is clone you gene to TA vector first, so you can do the sequencing very fast, then select the correct one and use the normal double digestion to ligate your gene to the desired vector.

Edited by Quasimondo, 07 March 2011 - 12:41 AM.


#4 muruga

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Posted 14 March 2011 - 01:42 AM

am doing transformation continuously for more than month. vector i choose is pET15b which is about 5.7kb my product size is 850bp. am using BamH1 and Nco1 as the restriction enzyme., For ligation reaction i use T4 ligase from sigma at 2 different conditions 4 and 22 C for overnight. Competant cells are prepared manually and cell pellets(ligated products+competant cells) of 100ul were into account for ligation. they were plated on amp plates. After overnight incubation colonies grown on the plates were taken for plasmid isolation but my product was not there wat may be the reason




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