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M_shabani_medbiotech

Member Since 03 Feb 2013
Offline Last Active Jun 10 2013 08:30 AM

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Group Active Members
Active Posts 12
Profile Views 419
Member Title member
Age 24 years old
Birthday January 10, 1989
Gender
Female

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My research interests
recombinant DNA technology

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Posts I've Made

In Topic: adding Tween80 to bacterial culture for protein purification ?!

10 June 2013 - 08:09 AM

actually i added it to culture media Posted Image

2 hours after i had added IPTG . tomorrow i'm going to perform ELIZA to detect my recombinant protein ! OH GOD , have i screwed up ?!! Posted Image

In Topic: plasmid extraction troubleshooting,please help !!

03 June 2013 - 06:18 AM

WOW ! u have A LOT of information !!

Thank u

potassim acetate is 3M . i don't know the PH of phenol chlorophorm . i should probably try 2.5 volumes of ethanol this time .

hey would u answer some other questions please ?
1. what exactly does potassium acetate do ?
2. what about i use isopropanol instead of ethanol ?

In Topic: plasmid extraction troubleshooting,please help !!

02 June 2013 - 09:40 AM

u know beside the low yield of plasmid concentration , another annoying problem i face is the huge amount of RNA even after Rnase treatment ! i would appreciate any kind of help with this problem too Posted Image

BTW , it's transformed into DH5a !

In Topic: plasmid extraction troubleshooting,please help !!

02 June 2013 - 09:35 AM

here is how i do it ...

1. Grow culture overnight in 5 mL of LB with antibiotic for 12-16 hours at 37C with 180 rpm shaking ( ampicillin 100 microgram/microlitr)
2. centrifuge 5 mL for 5 minutes and remove all media
3. Resuspend pellet in 200 uL GTE and let sit for 15 minutes
4. Add 400 uL if fresh 0.2N NaOH/1%SDS, mix by inverting 6 times, and let sit for 5 min on ice
5. Add 300 uL Pottasium acetate, and mix by inverting 6 times and let sit on ice for another 5 min
6. Centrifuge for 10 minutes
7. Transfer supernatant to new tube and extract with an equal volume of choroform, phenol, isoamyl alcohol
8. Vortex and centrifuge for 5 minutes
9. Transfer supernatant to a new tube
10. Add 2 volumes of cold 95% EtOH and leavein -20 for at least 1 hour
11.Centrifuge for 15 minutes at 4C
12. Remove supernatan and drain tubes on a paper towel by propping them up on a sharpie to allow the EtOH to drain off
13. add 1 mL cold 70% EtOH, and centrifuge for 5 minutes
14. Remove supernatant, drain well
15. Resuspend in 50 uL of TE..

In Topic: plasmid extraction troubleshooting,please help !!

02 June 2013 - 09:17 AM

oh right ! i'm sorry

i do alkaline miniprep , u know the protocol , right ?

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