Cloning from scratch, please help... - (Apr/06/2005 )

Dear all:

I'm a newbie in the lab and my advisor has thrown upon me a project for which I really have no clue as where to start...

My task is to clone cDNA of a receptor (named FceRI, high affinity receptor for IgE) from a cell line, RBL-2H3 (a rat basophilic leukemia that expresses tons of FceRI as I was told). This receptor was a tetramer (composed of alpha-beta2-gamma chains), whose cDNA sequences are obtainable from NCBI's nucleotide databases.

I thought about proceeding as follows, but wasn't sure if my logics was correct. Would be greteful if any expert could help correct my line of thoughts?

1. Isolate total RNA (or mRNA?) from RBL-2H3.
2. Do a reverse transcription (using oligo dT as primer?) to get cDNAs from mRNAs.
3. For each subunit, use the cDNA info (from NCBI) on the 5' direct and 5' complementary strands as sources of my sense/antisense primers; add any restriction site to the 5' end of each primer if necessary.
4. PCR to amplify cDNA for each subunit.


To be frank, I'm not sure if my reasoning on steps 2 and 3 above are accurate or not.
Many many thanks for any of your input in advance!!

hi, generally your strategy is okay. you should just assure that the ncbi sequences are full-length i.e. that you're not missing an upstream exon/atg or that there is no alternative splicing for your genes. to your other questions...

1. Isolate total RNA (or mRNA?) from RBL-2H3.

total rna is fine for your purposes; i usually use trizol (invitrogen) or tri-reagent (sigma) for this purpose

2. Do a reverse transcription (using oligo dT as primer?) to get cDNAs from mRNAs.

yes, use oligodT for 1st strand cdna synthesis; follow the instructions provided with your enzyme of choice, e.g. superscriptii from invitrogen

3. For each subunit, use the cDNA info (from NCBI) on the 5' direct and 5' complementary strands as sources of my sense/antisense primers; add any restriction site to the 5' end of each primer if necessary.
4. PCR to amplify cDNA for each subunit.

yes, exactly. but you may find it difficult to design "good primers" that amplify the cdna from start to stop, depending on the sequence context and your further downstream applications;

after 1st strand synthesis, take an aliquot of your 1st strand rxn, and set up your pcr; oligos should be ~25-27nt (if you have no other option to check the quality of your oligos, check out netprimer (premier biosoft), which kind of gives good estimates on the quality of your primers; this length could maybe also allow you to use a high annealing temp (~64-68°) which increases specificity obviously - of course this depends on your options for primer design)

you only need to add restriction sites to the primers if you dont directly clone your cdnas into t/a cloning vectors such as topo-ta vectors from invitrogen; dont forget to add some extra bases 5' to you restriction sites since some enzyme dont cut efficiently at the end of your pcr product (e.g. check the fermentas page http://www.fermentas.com/techinfo/re/index.html for this)

hope that helps a little bit. good luck.

Dear zybrg:

Couldn't say how much I appreciated your kind and detailed explanation on this for a newbie like me... Big thanks!!

One more question on this: I had read somewhere else that people also used "random primers" to get the first-strand cDNA from the total RNAs. Do I need to do so in my case? If so, how should I design those random primers? Thanks again!! :-)