chloramphenicol. - what is going on now?! (Sep/14/2006 )

kathy, i've used this method myself and that goes well with these concentrations. So i'm confident you can try it without going "directly in the wall". but i'm wondering : if the plasmid holds the chloramphenicol resistance, would this method be ok for you?...

pernesblue : i've tried to amplify a low copy plasmid in small cultures and the recovery was poor. Let say roughly 100µg for 50ml culture. So iswithce for 200ml culture and wasn't able to completelely get rid of RNA (so can't use it for my purposes), and columns were also partially blocked by over RNA so the recovery was equal.
I switched then for method described above, and the recovery was 450µg.

i've done plasmapper on pGEX 4T-1 sequence available on GE healthcare site...

Seems there is no chloramphenicol resistance holded by that plasmid...



Xiaoli Dong, Paul Stothard, Ian J. Forsythe, and David S. Wishart "PlasMapper: a web server for drawing and auto-annotating plasmid maps" Nucleic Acids Res. 2004 Jul 1;32(Web Server issue):W660-4.

here is the result :
LOCUS dna 4969 bp
FEATURES Location/Qualifiers
Promoter 184..212
/gene="tac prom"
Tag 258..921
/gene="GST tag"
ORF 258..977
/sequence="ORF_1 rf(3)"
misc_binding 930..935
/dbxref="REBASE:BamHI"
misc_binding 939..944
/dbxref="REBASE:EcoRI"
misc_binding 944..949
/dbxref="REBASE:XmaI"
misc_binding 944..949
/dbxref="REBASE:SmaI"
misc_binding 949..954
/dbxref="REBASE:AccI"
misc_binding 954..959
/dbxref="REBASE:XhoI"
misc_binding 959..966
/dbxref="REBASE:NotI"
Promoter 1307..1335
/gene="amp prom"
Marker 1377..2237
/gene="amp marker"
ORF 1377..2237
/sequence="ORF_2 rf(3)"
misc_binding 1918..1923
/dbxref="REBASE:PstI"
Rep_Origin 2392..3011
/gene="pBR322 origin"
misc_binding 2638..2646
/dbxref="REBASE:AlwNI"
Regulatory_Seq 3309..4400
/gene="lacI reg"
ORF 3441..4400
/sequence="ORF_3 rf(3)"
misc_binding 3875..3880
/dbxref="REBASE:ApaI"
misc_binding 4116..4121
/dbxref="REBASE:EcoRV"
misc_binding 4172..4177
/dbxref="REBASE:HpaI"
misc_binding 4307..4312
/dbxref="REBASE:NarI"
Promoter 4449..4478
/gene="lac prom"
Reporter 4540..4699
/gene="lacZ_a reporter"
BASE COUNT 1225 a 1202 c 1292 g 1250 t 0 others
ORIGIN
1 acgttatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc ggaagctgtg
61 gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc gcactcccgt
121 tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc tgaaatgagc
181 tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca
241 cacaggaaac agtattcatg tcccctatac taggttattg gaaaattaag ggccttgtgc
301 aacccactcg acttcttttg gaatatcttg aagaaaaata tgaagagcat ttgtatgagc
361 gcgatgaagg tgataaatgg cgaaacaaaa agtttgaatt gggtttggag tttcccaatc
421 ttccttatta tattgatggt gatgttaaat taacacagtc tatggccatc atacgttata
481 tagctgacaa gcacaacatg ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc
541 ttgaaggagc ggttttggat attagatacg gtgtttcgag aattgcatat agtaaagact
601 ttgaaactct caaagttgat tttcttagca agctacctga aatgctgaaa atgttcgaag
661 atcgtttatg tcataaaaca tatttaaatg gtgatcatgt aacccatcct gacttcatgt
721 tgtatgacgc tcttgatgtt gttttataca tggacccaat gtgcctggat gcgttcccaa
781 aattagtttg ttttaaaaaa cgtattgaag ctatcccaca aattgataag tacttgaaat
841 ccagcaagta tatagcatgg cctttgcagg gctggcaagc cacgtttggt ggtggcgacc
901 atcctccaaa atcggatctg gttccgcgtg gatccccgga attcccgggt cgactcgagc
961 ggccgcatcg tgactgactg acgatctgcc tcgcgcgttt cggtgatgac ggtgaaaacc
1021 tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca
1081 gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc
1141 agtcacgtag cgatagcgga gtgtataatt cttgaagacg aaagggcctc gtgatacgcc
1201 tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc
1261 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
1321 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
1381 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
1441 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
1501 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
1561 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgtg
1621 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
1681 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
1741 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
1801 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
1861 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
1921 cagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
1981 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
2041 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
2101 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
2161 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
2221 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
2281 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
2341 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
2401 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
2461 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
2521 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
2581 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
2641 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
2701 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
2761 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
2821 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
2881 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
2941 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
3001 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
3061 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
3121 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
3181 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc ataaattccg
3241 acaccatcga atggtgcaaa acctttcgcg gtatggcatg atagcgcccg gaagagagtc
3301 aattcagggt ggtgaatgtg aaaccagtaa cgttatacga tgtcgcagag tatgccggtg
3361 tctcttatca gaccgtttcc cgcgtggtga accaggccag ccacgtttct gcgaaaacgc
3421 gggaaaaagt ggaagcggcg atggcggagc tgaattacat tcccaaccgc gtggcacaac
3481 aactggcggg caaacagtcg ttgctgattg gcgttgccac ctccagtctg gccctgcacg
3541 cgccgtcgca aattgtcgcg gcgattaaat ctcgcgccga tcaactgggt gccagcgtgg
3601 tggtgtcgat ggtagaacga agcggcgtcg aagcctgtaa agcggcggtg cacaatcttc
3661 tcgcgcaacg cgtcagtggg ctgatcatta actatccgct ggatgaccag gatgccattg
3721 ctgtggaagc tgcctgcact aatgttccgg cgttatttct tgatgtctct gaccagacac
3781 ccatcaacag tattattttc tcccatgaag acggtacgcg actgggcgtg gagcatctgg
3841 tcgcattggg tcaccagcaa atcgcgctgt tagcgggccc attaagttct gtctcggcgc
3901 gtctgcgtct ggctggctgg cataaatatc tcactcgcaa tcaaattcag ccgatagcgg
3961 aacgggaagg cgactggagt gccatgtccg gttttcaaca aaccatgcaa atgctgaatg
4021 agggcatcgt tcccactgcg atgctggttg ccaacgatca gatggcgctg ggcgcaatgc
4081 gcgccattac cgagtccggg ctgcgcgttg gtgcggatat ctcggtagtg ggatacgacg
4141 ataccgaaga cagctcatgt tatatcccgc cgttaaccac catcaaacag gattttcgcc
4201 tgctggggca aaccagcgtg gaccgcttgc tgcaactctc tcagggccag gcggtgaagg
4261 gcaatcagct gttgcccgtc tcactggtga aaagaaaaac caccctggcg cccaatacgc
4321 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc
4381 gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca
4441 ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa
4501 caatttcaca caggaaacag ctatgaccat gattacggat tcactggccg tcgttttaca
4561 acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc
4621 tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg
4681 cagcctgaat ggcgaatggc gctttgcctg gtttccggca ccagaagcgg tgccggaaag
4741 ctggctggag tgcgatcttc ctgaggccga tactgtcgtc gtcccctcaa actggcagat
4801 gcacggttac gatgcgccca tctacaccaa cgtaacctat cccattacgg tcaatccgcc
4861 gtttgttccc acggagaatc cgacgggttg ttactcgctc acatttaatg ttgatgaaag
4921 ctggctacag gaaggccaga cgcgaattat ttttgatggc gttggaatt
//

Fred, thank you so much for your help. In the molecular cloning (from where i decided the concentration of Cm) it says that pBR322 origin plasmids treated with 10-20ug/ml of Cm have improved plasmid yields. i think resistance to Cm is not accurate term and i am sorry i lead everyone into confusion. maybe it is that Cm has good effect only on low copy plasmids but not high copy plasmids. so i guess my plasmid (even if it doesnt have Cm resistance) should be effected by Cm treatment. and if authors say so especially. but noone says culture on Cm plate. what do you think?

I guess it is simple enough. The plasmid gives AmpR, it doesn't give chloramphenicol resistence. So Cm can not be used in the normal growing phase. All plating and culturing work must be done using Amp.

As Fred has described earlier chloramphenicol simply blocks cells growth (at the concentrations used) while allowing plasmid DNA synthesis to continue. Thus allowing plasmid number to build up.

This step can be done, or you could just grow more cells, maybe 500ml or a 1L if required. And forget about the Chloramphenicol induction.

yep, agree with pernesblue.
But culturing great volumes gives pb in purification. The RNA is very high, and so is difficult to get rid of or to prepare in columns.
In that case you may consider a CsCl gradient for better prufication.

thanx a lot guys! i have grown my cells in the absence of Cm and i got my protein... so i guess it was not so necessary from the first place. PIs always panic dont they....

sincere congratulations kathy.

Hi all. I´m not sure if that´s the topic, but at least in our protocol, the chloramphenicol resistance is carried by the cell strain (BL21 LysS) (for expression of recombinant proteins) and not by our protein fussion plasmid. So you select for both your construct plasmid and the T7 polymerase "endogenous" plasmid responsive to the induction step. When we grow for plasmid prep and not for protein expression, we only select for Amp resistance. Chloramphenicol is not strictly necessary in our hands, but if you use it is better to do it from the plating step. so Fred, you use chloramphenicol in "non-resistant" bacteria to block metabolism? Is that ok?

Hope it helps.

i need toclarify my post.
When growing non Cm-resistant bacterias, either with a Cm-gene-containing plasmid or not, i do not select by amp and cm at the plate step.
I do chloramphenicol selection when preparing bunch of LOW copy plasmid. The effect of chloramphenicol is to "enrich" the culture with plasmids and reduce the RNA.
So the chlramphenicol step is not suitable for cm resistant strain and/or for Cm-resistance-gene containing plasmids.