28 replies to this topic

[Fluffy]

Hello again,

So I did my step 1 of my protocol yesterday (transformation and plating). The plates have different plating volumes (100uL, 200uL, 300uL, and 400uL). I am not sure if my transformation worked the way it should. I mean I have ALOT of small colonies on my plates. So do you think I should move on to step 2 today? And if so, do I choose any single colony from any plate and inoculate that? Please respond back to me on this.

Thanks again :)

[John Forsberg]

When you're transforming bacteria from purified plasmid DNA, you should get tons of colonies (unless your DNA or cells are poor quality).

There shouldn't be too much colony-to-colony variability in expression, so you can pick any colony and it should be okay.  I usually inoculate 2 colonies in 2 tubes whenever doing a new growth in case one of them doesn't grow (for whatever reason).  I would avoid any colonies that look significantly different from the others (may be a sign of contamination).

[Fluffy]

Thanks John That actually made me feel better about my transformation. Okay I will do just like you then, I will inoculate two colonies. Thanks for the hints.

All the best to you

[Fluffy]

I am still confused about OD600 measurement, and I am worried that I did it wrong last night.

I will clearly indicate my steps. I am using a cuvette spec.

For blank, I took 1ml fresh medium and diluted by adding 3ml water
For bacterial culture samples, I took 1ml of bacterial culture and diluted by adding 3ml water as well.

The cuvettes I am using are ones that light beam can pass through all sides. I made sure it is clean.
I loaded 1ml of my diluted samples in the cuvettes.

I blanked the machine and Abs. was zero.
I measured my diluted samples which I got negative Abs. for them

Looking at the cuvettes it seemed like the 1ml was not enough and I thought to myself that light is possibly not going through the solution at all to give a reading.

I ended up doing it again by filling cuvettes with 3ml of the diluted blank and the diluted bacterial culture samples (prepared above).
For blank I got zero.
For samples since I had two differet samples, one I got Abs. 0.321 which I multiplied by 3 (dilution factor)= 0.963, and the second one I got an Abs. of 0.360 which I also multiplied by 3 (dilution factor)= 1.08

Based on these Abs. I moved on to the induction step.

But I was thinking about it the whole night and I have a feeling that I am not correctly measuring OD600.

Please help me out, I am stressed about this.

Thanks all

Edited by Fluffy, 16 November 2012 - 06:50 AM.

[Fluffy]

Sorry in addition to the question I posted earlier about OD600 readings, I also have another question about the future steps. I remember in one of the posts Andreea mentioned that after a successful induction I can spin down (centrifuge) my culture to obtain pellets that can be stored at -80C until analysis. For analysis I wanted to compare the total cell protein fraction in parallel with other fractions such as the medium fraction, soluble cytoplasmic fraction, and the insoluble cytoplasmic fraction for the recovery of my target gene. In the pET manual 11th edition, there are protocols on how to isolate those fractions and then analyze them by SDS-PAGE. I wanted to follow their protocols since they are well written and explained but the problem is I won't have time to do it on the same day. I would have to as Andreea mentioned spin down the cells and store the pellet at -80C. If that ends up to be my case, can I still use my stored pellet to follow their protocols in the manual. I am worried since in their manuals, the protocols for the extraction of the fractions mentioned above always have as their first step: Prior to harvest take a 1ml aliquot of the culture..etc..
And this is not my case since I harvested the cells (pellet at -80C). What do you think I would have to do?
I hope I was clear enough I am sorry if this is confusing.
Thanks

Edited by Fluffy, 16 November 2012 - 05:12 PM.

[ascacioc]

For the first part: I usually measure undiluted, but it works like you too; actually, if you are trying to get OD600 = 1 then you should dilute. I induce myself at 0.6
For the second part: you can keep your pellets also at -20 (in case you need space in your -80; I surely always have this problem)
I always resuspend the pellet in 5 mL of buffer and take 100 uL of this + 50 uL 3X loading buffer (load only 3 uL on a gel because it is very concentrated); sonicate these 5 mL and spin down for 30 min at the max speed of the centrifuge I have at 4oC; take 100 from supernatant and treat as above; from the pellet, resuspend it in 2 mL of water and take 100 uL and treat as above; the rest of the 5 mL of the soluble fraction I incubate with 20 uL affinity beads for my tag on rotation in the cold room for 1-2 h and then wash 3-5 times and then boil the beads in sample buffer and load 10 uL of this on a gel.

Andreea

[Fluffy]

Andreea I am not sure about the second part. I am sort of confused as to how you do it. But can I follow the pET manual protocols even if I centrifuged down my culture and stored as a pellet?
I guess I can since I centrifuged down 1ml culture aliquots into pellets and stored at -80C. In the pET manual it says to take 1ml aliquots and work with those. I am just worried because I stored my pellet and it does not mention to store the pellet in their protocols.

[ascacioc]

it is ok with frozen pellets as well

[ascacioc]

BTW: when you harvest your cells for purification you can also freeze the pellets before you start the purification which will take the entire day

[Fluffy]

Hello I am back

I did an SDS-PAGE that included total cell protein fraction, cytoplasmic soluble fraction, and cytoplasmic insoluble to test the expression.
My ladder is suppose to have a pink orientation band at 64kDA. The link to it is http://products.invi...roduct/10748010 But for some reason the pink band did not appear on my ladder so it is kind of hard for me to figure out my protein band. My protein size is ~16.52kDA.

The organization of my SDS-PAGE image below is
far left-ladder, uninduced total cell protein, induced total cell protein, uninduced cytoplasmic soluble, induced cytoplasmic soluble, uninduced cytoplasmic insoluble, induced cytoplasmic insoluble-far right.




It is kind of hard for me to distinguish my protein band..any suggestions as to which is mine? Also if it is there is my expression good, can I go on?

As you can see also the insoluble fraction shows nothing at all?? Is that normal?

Please provide me with some feedback.
Thanks All

Edited by Fluffy, 01 December 2012 - 11:33 AM.

[ascacioc]

I am also confused myself. Is that a 15% gel? I guess it must be if you want to see your protein of 16 kDa Anyhow, what I can tell you is that sometimes in the uninduced fractions you could also see your protein when you have leaky expression which can happen as T7 from pET system is a very strong promoter and it is very difficult to inhibit before expression is induced. I would do this experiment with the beads that I have suggested above, like this you can observe whether the enriched fraction contains a protein around your size. I would guess that the pink band did not separate enough and that you have from bottom to top: 6, 15 and so on and the top marker bands that run all together contain the pink band as well. I would not continue until I see the enriched band on the gel from the beads experiment. However, you could also try direct large scale expression. Most people around me are lucky enough for it to work from the first trial

Andreea

[Fluffy]

Thanks for your response Andreea. Hmm...when you say beads you are referring to purification step correct, then run the eluted sample on SDS-PAGE? I am planning on doing that as well but I am waiting on a kit on order. And this would most likely be a really important step and what my professor would like to see. He wants to see if possible what other proteins interact with my protein of interest after I purify my his-tagged proteins using a column and run the eluted sample on SDS-PAGE gel. According to him that if any proteins do interact with my his-tagged protein they would get pulled down (eluted sample), and should appear as well on the SDS-PAGE along with my his-tagged protein. I am looking forward to that step.

I am not sure though if I will be using the same beads as you for purification. My professor will order His•Bind Purification Kit by Novagen. It is compatible with the Popculture Reagent and the BugBuster Master mix reagent that I used to extract my soluble and insoluble fractions.
I guess that should still be fine. Also another question, the past cell paste extractions that I used to run my SDS-PAGE gels are all used up. I have more cell pellets stored from the same induction experiment, it would be fine to do other round of total cell protein, soluble, insoluble protein extractions and use those extracts for my purification step? I mean the cell pellets are from the same culture I just aliquoted them in epp. tubes and harvested their cell pellets.

Sorry I ask too many questions this forum is my only source of support

Thanks Again

[ascacioc]

Sorry for taking me ages to reply. I have been on vacation.

beads experiment: take the pellet from 50 mL of culture, resuspend in 5 mL of buffer, sonicate, spin at highest centrifugation speed your table centrifuge allows (at 4oC) and then incubate 2 mL of supernatant for ~2h with 20 uL of affinity beads (equilibrated in the proper buffer). Then wash the beads 3-5 times with the buffer while centrifugating at low speed for 2 min in between washes. Load this on the SDS-PAGE.

Interaction partners: well, it might not work as your professor thinks. You need a very stable interaction and if you have a transient interaction as in regulatory pathways, these are not stable complexes (trust me, I also have the nightmare of transient complexes in my PhD thesis). Moreover, even if you have a stable interaction, if you have more than 150 mM salt (NaCl or KCl) in the binding buffer, this complex might disassemble. Furthermore, if you have to elute the protein from the column, you need to add imidazole, which is a salt and will increase the total amount of salt in the buffer so your interaction partner will dissociate before your his-tagged protein.

Your frozen pellets are good forever...ok not forever, but until the end of your project as long as it does not last 10 years. So, yes you can use the ones you have in the freezer.

Andreea

[Fluffy]

Hello Andreea,

Oh nice hope your vacation went well. It is always good to have rest. I took a vacation too . Thanks for getting back to me dear. I will take your ideas into consideration for the next steps of my work. I recently ran a western to confirm my protein sizes. Before I post my pictures to get you to look at them. I have a couple of questions, I want to make sure I am calculating my protein sizes in kDA correctly.

First off, as you remember I have two genes that I work on and expressed. My genes are UBC3 and UBC1.

UBC3 cDNA sequence + cloning primers
    1 ctcttcttcc atttctttca aaattaaagt attgttactc tgctattggc tcaaaacctc
   61 tgcaatctcc gtctccttca atttcaactc aagcaaatcc acctctttca ctagtttcat
  121 cactttcaga tcagggtttg gagttgaagg tacggggggc taattgatgg cgtcgaagag
  181 gatattgaag gagctcaagg atctgcagaa ggatcccccc acatcatgca gtgctggtcc
  241 agtggcagag gatatgttcc attggcaagc aacaatcatg gggcctaccg atagccctta
  301 tgctggaggt gtatttttgg tttcaattca tttccctcca gattatcctt ttaagcctcc
  361 aaaggttgcc ttcagaacta aggttttcca tcccaacatc aacagcaatg gaagtatttg
  421 tctggatatt cttaaggagc agtggagtcc agcattaacc atatccaagg tcctgctgtc
  481 catctgctct ctgttgacag acccaaaccc agatgatcct cttgtacctg aaattgctca
  541 catgtacaag actgacaggg ccaaatacga aaccactgct cgtagctgga ctcagaaata
  601 tgcaatggga tgatgcgcaa aatgtctcca ggcatgtctg ggactttgta acagcaatgt
  661 cttatgtgct tggggtgaat gaataaattc cgtgaaagaa cttagttact tcttaatctc
  721 ccttcatgag ggttgttaag ggaacagctg ttttcaattt gtgaatattt atttgatgac
  781 tagtaaggga gaaactgcaa tgtaattcta ctttgtttgc cagtt
Primers
Forward Primer (NdeI)
5’ c a t a t g a t g g c g t c g a a g a g g a t a t t 3’
Reverse Primer (XhoI)
5’ c t c g a g t c a t c c c a t t g c a t a t t t c t g 3’
UBC1 cDNA sequence + cloning primers
    1 gcacgagggc gacttttgca taaaccaaaa ttagaatcaa attggaagag agaaaaaaaa
   61 tggtggactt ggctagggtt caaaaggagc tccatgaatg caacagagat gttcaggttt
  121 ctggaattaa tgttaccctt aaaggtgaca gtctcactca cttgattggt acaatccctg
  181 gtcctgttgg tactccttac gaaggcggta ctttcaagat cgatatcact cttactgatg
  241 gctacccatt tgagcctcca aaaatgaaat tcgccacaaa agtttggcat cccaacataa
  301 gtagtcaaag tggagcaata tgcctagaca tcctgaagga ccagtggagc ccagcactaa
  361 ctctcaagac agctctcctt tctatacaag cattactttc tgctcctgaa cctgatgatc
  421 cacaagatgc agttgttgca cagcagtatc ttagagaaca tcagaccttt gtcggcacag
  481 ctcgttactg gactgagact tttgcaaaaa catccacact tgctgcagac gacaagatac
  541 aaaagcttgt ggaaatgggc tttcctgaag ctcaagtgag gagtactttg gaagcaaatg
  601 gttgggatga aaacatggct cttgaaaagc tgttgtccag ctaaaaccct tctactgcaa
  661 ctcatatttt gataagacaa ttatatcctt ccagcaaaag ctgatgacta gaatagagtc
  721 actcggttat actgttgctt ggcaatcttg tttctgtctc ctttatggtt tgctgttgac
  781 atctcttcat atcctgtgaa gattctgatg ttatttttac aatatcaagc aaattgcata
  841 tgaatcatgg ggaggaagtg gactttccgg ggtgaaaaaa aaaaaaaaaa aaaaaaaaaa
  901 aaaaaaaaaa aaaaaaaaaa aaa

Primers
Forward Primer (NdeI)
5’ c a t a t g a t g g t g g a c t t g g c t a g g g t 3’
Reverse Primer (XhoI)
5’ c t c g a g t t a g c t g g a c a a c a g c t t t t c a 3’

Using google I found protein translation tools, I translated the above cDNA sequences into protein sequence. Then using google search I used a protein size calculation tool to estimate protein sizes of the above two genes. My estimated protein sizes are 16.52kDA for UBC3 and 21.38kDA for UBC1. Please do you think these are accurate not necessarily perfect? I need to know the best estimated protein sizes for the two genes for my western blot please. Also for estimating the protein sizes for western do I need to put into consideration the size of the His-tag?

Thanks Andreea so much