Search found 259 matches

by biology_06er
Sun Oct 23, 2011 10:44 am
Forum: Molecular Biology
Topic: GFP
Replies: 7
Views: 3903

GFP

Hi there, So I recently ligated my gene into a Pet32a vector using restriction sites using EcoR1 and Xho1. From another Pet vector I have to create primers for the GFP insert containing BgII and EcoR1 sites. Just wondering, once I amplify my GFP (using BgII/EcoR1 sites I assume), how do I ligate it ...
by biology_06er
Tue Oct 11, 2011 2:01 am
Forum: General Discussion
Topic: MCAC
Replies: 1
Views: 1073

MCAC

Hi there,

Regarding protein purification I have to make up buffers
MCAC-0: 20mM Tris.HCl, pH7.9
0.5 M NaCl
10% glycerol

MCAC-1000: 20mM Tris.HCl, pH7.9
0.5 M NaCl
10% glycerol
1M imidazole

what does MCAC-0, 1000 mean. What does the number after it mean?
by biology_06er
Thu Oct 06, 2011 2:01 am
Forum: Microbiology
Topic: ligation of PCR product
Replies: 3
Views: 6121

Re: ligation of PCR product

n/m...I understand now!
by biology_06er
Wed Oct 05, 2011 11:15 pm
Forum: Microbiology
Topic: ligation of PCR product
Replies: 3
Views: 6121

Re: ligation of PCR product

I did also use the gel purified DNA but also was told to try ligating the pcr product straight into the vecotr...i have no idea what the "procedure" for this is..i have looked and looked for one online...was this part at least correct? Cut my vector with 1uLxho1 and 1uL ecor1 and correspon...
by biology_06er
Wed Oct 05, 2011 8:01 am
Forum: Microbiology
Topic: ligation of PCR product
Replies: 3
Views: 6121

ligation of PCR product

Hi there, Today I used my PCR product (that was cleaned up) to ligate into PET expression...just want to see if I went about it the right way: Cut my vector with xho1/ecor1 and corresponding buffer and water up to a volume of 20uL...kept at 37 degrees for around an hour cut my pcr product with same ...
by biology_06er
Sun Sep 25, 2011 3:10 am
Forum: General Discussion
Topic: overnight culture/minipreps
Replies: 4
Views: 5576

Also--when I did a single colony PCR, I streaked a fresh plate for each selected colony to see if my positives correlated with actual growth overnight (as well as for further bacterial stock for later use)...the postives that showed up on my gel did grow overnight but also the ones that were negativ...
by biology_06er
Sun Sep 25, 2011 1:29 am
Forum: General Discussion
Topic: overnight culture/minipreps
Replies: 4
Views: 5576

overnight culture/minipreps

Hi there, So for the last week and a bit ive been doing minipreps and when I run a gel I don't get a bright band as I should..I've picked every positive colony after running a single colony PCR and inoculatedeither 5mL or 10mL of LB-media each time (w/ ampicillin)...the other day I did a 50mL o/n cu...
by biology_06er
Fri Sep 16, 2011 7:40 am
Forum: General Discussion
Topic: syber safe dilution
Replies: 1
Views: 1713

syber safe dilution

Hi there,

I was making a 100mL volume of syber safe today using a starting conc. of 10,000x. Have I done the calc right if i wanted the end conc to be 1x?..

1*100/10000=0.01mL=10uL..so added 10uL of 10,000x stain to 100mL of 1x TAE buffer?

Was this correct?
by biology_06er
Wed Sep 07, 2011 7:17 am
Forum: General Discussion
Topic: how long does bacteria last
Replies: 1
Views: 1737

how long does bacteria last

Hi there, On friday (2nd sep) I carried out a single colony PCR and streaked a new plate with the colonies I selected...I kept it overnight and put the plate in the fridge sat morn...yesterday (monday) I did an overnight culture on one of the colonies and today made a glycerol stock of it...but I I ...
by biology_06er
Thu Aug 18, 2011 2:48 am
Forum: General Discussion
Topic: plasmid cut and uncut
Replies: 2
Views: 4194

plasmid cut and uncut

Hi there,

I have to bands on my gel..one uncut plasmid and one cut...they were more or less the same size with the cut being slightlyyy bigger..(looking at them both compared to the ladder)....Just wondering if there is a reason for this?
by biology_06er
Mon Aug 15, 2011 11:35 am
Forum: General Discussion
Topic: mature form of a gene via PCR
Replies: 2
Views: 2112

mature form of a gene via PCR

Hey guys, So I have recently PCRed my complete gene of interest/carried out blue white selection and the next step (well not next step but something I will have to do very soon) is to PCR the mature form of the gene..so what is the general process here..from genomic DNA, I used primers etc and got a...
by biology_06er
Fri Aug 05, 2011 11:55 am
Forum: General Discussion
Topic: how to work out how much IPTG/X-gal for plating
Replies: 1
Views: 3051

how to work out how much IPTG/X-gal for plating

Hi there, The other day I was preparing agar plates for the first time and today I had to redo it using a slightly different method regarding adding the IPTG/X-gal. The first time I was told to separately add 5uL of IPTG and 50uL of X-gal to each plate and spread it but the second time to make it a ...