I have been working with screening libraries of cDNA prepared in Lambda ZAP-II phages of Plasmodium falciparum. After screening we found 5 positive clones, reactive with monoclonal antibodies. We need to prepare a sample for DNA to get sequences of gene codifying proteins reactive with the antibody. We think the better option at this moment is to obtain cDNA inserts or the phagemids infected with M13 helper phage and after in vivo excision get the phagemid using to plating the fphagemids using the E. coli SORL strain. Does anybody have helper phage M13 and the SORL strain and can help me?
Yes, Gladys, you are right. Lambda ZAP libraries can be "excised" to recover the insert in the form of plasmid. This is much more convenient than mini-preping and sequencing lambda clones. Unfortunately I can not provide you with these reagents (it's been years since I finished using lambda libraries) but you can try to buy them from Stratagene-Agilent, they have a "Rapid Excision Kit" with improved reagents (fagemids and E.coli strains).
Finally my congratulations for having positive clones.Screening lambda libraries with antibodies is a tough, very difficult work!! I hope that you are lucky and get your antigen.
Disclaimer, no relationship with Stratagene-Agilent other than user of their products
Are the inserts large? If they are relatively short, you could try skipping the entire DNA purification/excision altogether by amplifying directly from lysogens/phage suspensions by PCR with some long range enzyme mix and universal primers, then sending the amplicons to your sequencing facility.
Thanks Estanis
Alejandro: I don´t know how big our inserts are. I understand that independienty how big are genes, inserts could be smallers than complete gene. It depends on the cDNA library preparation. Alejandro you are rigth we can amplify the inserts using primers as T3 -T7. However, need first obtain excised phages as phagemids and amplify the inserts using those primers and send as amplicons to sequencing.
You do not need to excise the phagemid to PCR the inserts out; if I remember correctly in lambda ZAP what is split is the M13 ori, right? The entire MCS region (and flanking promoters) will be contiguous. You can use directly lysogenic colonies (a la colony PCR) or lambda phage lysates, assuming that you can go either way with lambda ZAP.
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