progress update (Oct)

KAS I

i got a new PCR primer sequence because before this, there seems to be some problems with the primers we ordered. so, once i got a new primer, i start all over again from PCR, cloning and transformation. i manage to get the gene KAS I into pQE30 plasmid and transform it into E.coli M15 and JM109 host. after i sent the plasmid for sequencing, luckily, there's no problem anymore. the sequence are okay. so now i finished with my first part. for my second part, i will start to express my KAS I protein in both E.coli JM109 host and M15 host. further progress will be updated soon.

-HANI-

progress report 2

29/7/2010

After we analyzed the sequencing results, actually there were some problems.

KAS I: - we manage to get the correct insert (KAS I) in pQE30. however, when we analyze the nucleotides sequence, one of the nucleotide was missing at the primer sequence. so, we already sent the same plasmid for re-sequencing for confirmation. there are 2 probabilities; first, problems maybe occured during the sequencing process, or problems with the KAS I primer which the BioNeer did not give the complete primer sequence.

KAS II: - basically we manage to get the correct insert (KAS II) into pQE30. The problems also the same as KAS I, but, instead of missing one nucleotide, it happen to be an extra nucleotide at the primer. so, for confirmation, we decided to send for re-sequencing again and compare with other clones.

KAS III : - we not able to sequence this pQE30+KAS III because we got the wrong clone. maybe KAS III was inside the pGEMT vector. thus, we planned to digest the pGEMT-KAS III and ligate it with pQE30 into E.coli JM109 again and send for sequencing.

That's all.

More progress will be updated soon.

-HADI /HANI-

progress updated!

(19-24 July 2010)

last monday, we sent our plasmid extraction samples (pQE30+KAS in E.coli JM109) for sequencing.

out of KAS I and KAS II, both samples gave positive result.

only KAS III gave negative result. according to First Base, they able to sequence the forward primer of pQE30+KAS III, but unfortunately they cannot sequence for the reverse primer for pQE30+KAS III. i'm not sure what's the problem. but we will send pQE30+KAS III for sequencing again. this time we try to extract a different samples and send for sequencing.

when we have confirm the sequencing result, we will proceed with transformation of pQE30+KAS into E.coli M15 and protein expression.

we try to express pQE30+KAS using two different hosts.

pQE30+KAS in E.coli JM109, and also pQE30+KAS in E.coli M15.

that's all.
will update more...
thank you.