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kingswill's Content

There have been 11 items by kingswill (Search limited from 03-April 19)


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#107836 Number of publications after PhD

Posted by kingswill on 24 April 2011 - 07:00 AM in Career Advice

I had zero at the time when I got my first postdoc offer.



#97064 Acrylamide stock

Posted by kingswill on 10 January 2011 - 06:38 AM in SDS-PAGE and Western Blotting

What do you mean by extra? Is it the solution left behind after you pour your gel? Then is your gel ok? If you gel is ok it's not a problem I suppose?

If you gel is not set, first make sure you mix the solution gently to minimize air in your solution (as bob stated the reason), or you may go for de-gasing (Never done this before but if gas is a real problem) . Then check your APS (this is the most likely part that causing un-set gel), prepare a new 10%APS if needed. Once I found my APS powder looked hydrated and my gel didn't set even I prepared APS solution fresh. I borrowed APS powder from other and it worked fine. Then check Temed and Acrylamide.



#97062 What cause "downshift" in SDS-PAGE?

Posted by kingswill on 10 January 2011 - 06:13 AM in SDS-PAGE and Western Blotting

Actually itīs phosphorylation, not dephosphorylation that causes proteins to migrate faster.


not to my understanding. Phosphorylation should cause a upshifted band.



#97043 What cause "downshift" in SDS-PAGE?

Posted by kingswill on 09 January 2011 - 07:44 PM in SDS-PAGE and Western Blotting

There's a protein sample. You splite it into two and subject them to two treatment (presumably 1 control treatment and 1 testing treatment). After that you denature it and run SDS-PAGE. You find that the testing treatment "downshift" the protein, i.e. protein migrate faster than the control. What is/are the possible causes of this?

Everybody knows the most obvious one is phosphatase (or dephosphrylation). I just wanna if there's any possbile mechanism to cause this observation.



#93946 Site directed mutagenesis

Posted by kingswill on 03 December 2010 - 01:09 PM in Molecular Biology

Hello!
I'm doing site directed mutagenesis to point mutate one bp using Phusion SDM kite from Finnzyme. I did the PCR and run a bit on gel to check. The product looks smaller than it should be and I'm a bit concerned now and would like to ask anybody here has experience in using this kit or SDM in general. Let me give some more details:

Source of template: miniprep plasmid
Primers: 5'phosphrylated and RP-HPLC purified (a must for this kit). double checked (in silico) they matched the regions I wanna mutate and don't cross-hybridize to any other region of the gene or in the backbone.
Expected product size (=size of the contruct): 6kb
PCR program: 98 10s, (98 10s, 72 3min30s) x 25, 72 10min (Tm is close to 72 (71.8), so the annealing and extension steps are combined. Extension time is based on the rate of 30s/kb)

After PCR I run the product on gel (1%). It appears around 4kb. I run alongside a linearized version of the same construct and it appear correctly at around 6kb. I have a control without Phu enzyme and there's nothing, which means that first template doesn't contribute to anything in the gel and second any bend in my sample should come from PCR amplification. I also have a positive control using a control plasmid provided by the kit which show a bend with correct size. This seems eliminating the possibility that anything eg salt and protein etc in the PCR reaction mix alter the migration pattern of the DNA in agarose gel.

I keep going to ligate the product and will transform them with a hope that the product is alright. However I drop my question here first in case I have to troubleshoot it later (fingercross I don't need to!)



#86861 Protein expression

Posted by kingswill on 14 September 2010 - 07:27 AM in Protein Expression and Purification

Protein ladder gives you an estimate of the size of your protein but not absolute. What % is your SDS-PAGE? You may try higher % gel to give you a better resolution of proteins as that size.

Another possibility is phosphorylation which may cause an upshift in your gel. Bacteria contain machineries that may phosphorylate your protein from human or whatever species. You can dephosphrylate you sample and see if the band shift back down.



#86860 Ligation problem: clones survive selection but nothing in there?

Posted by kingswill on 14 September 2010 - 07:10 AM in Molecular Biology

I and my lab transformed pCS2+ constructs to the same batch of competent cells many times before and we didn't encounter this problem. I guess if the backbone contains homologous sequence to the host genome then we should see this more frequently.

it looks more and more likely that I did some mistakes during the whole process may be adding the wrong buffer during mini? Let see what happen in my second trial.



#86732 Ligation problem: clones survive selection but nothing in there?

Posted by kingswill on 13 September 2010 - 07:19 AM in Molecular Biology

thanks!

My vector is pCS2+. It's supposed to be high copy.

I think I'll do the whole experiment again starting from transformation as I have ligation products left and see what happen. I'm still not sure what is/are the causes. About the low copy number and chromosomal integration, because there are four different fragments that I'm subcloning into pCS2+ and in total tens of clones were picked, grown and mini, it's highly unlucky, if not unlikely, for all of them to suffer the same problem.



#86712 Ligation problem: clones survive selection but nothing in there?

Posted by kingswill on 13 September 2010 - 03:05 AM in Molecular Biology

update

Spread three plates from three tubes of competent cells. Nothing is growing. May be it's that particular tube having problem?
Wanna make sure one thing. I thaw the cells and then spread them on plates directly. Was it ok? Because when I do transformation I heat shock them and then add LB without amp and let them grow up a bit for an hour before spreading on plate. I don't know if spreading them directly will affect viability.



#86566 Ligation problem: clones survive selection but nothing in there?

Posted by kingswill on 10 September 2010 - 05:04 PM in Molecular Biology

thanks for reply!

the idea of my competent cells somehow getting resistant did come across in my mind. Yes I should spread some and see what happen.
This lot of competent cells were prepare in bulk by a previous post doc in my lab, which are very good in previous transformation and cloning and I use them for many times. They are in small aliquot and we never re-freeze and throw away once an aliquot is thawed and used. I hope it was that particular tubes that a problematic otherwise I have to throw way the whole lot and prepare again.

Just for interest, how a tube of bacteria get resistant at -80?



#86550 Ligation problem: clones survive selection but nothing in there?

Posted by kingswill on 10 September 2010 - 12:32 PM in Molecular Biology

Hello!
I'm doing several ligation experiment, some are blunt-sticky and others are sticky-sticky (there are non-compatible). After ligation I transformed them into bacteria, spread them on plates, picked some, grew them in 1ml culture, mini, did restriction mapping and checked for insert.

When I run gel, I saw nothing. Blank. First I thought may be the yields were too low after mini. Confirmed by OD 260 that concentration in the mini elute were- very low, although in theory I should see them in gel (I use about 50ng-100ng for each restriction mapping).

Then I grew them up again (I kept 100 micro litre untouch from the each original 1ml culture) in 10ml culture hope to get higher yields. mini. OD260 told the yields were a bit higher than previous ones but still lower than normal mini yield. I decided carrying on the restriction mapping. Nothing in gel again. Blank.

Here is my thought:
1. mini kit may be defective. but I used this kit (I mean the columns and buffers from the same box of mini kit) many times before and they were fine. So I doubt.
2. my ampicilin in the agar plates and LB may go off. The plates were 3-4 weeks old stored at 4 degree which may be problematic. However I add fresh, whole new aliquot of Amp to the LB that I used to expand the culture. So I doubt.
3. Unless the whole lot of amp in my -20 freeze has gone off already. This could explain why I saw bacteria growing and yet got nothing from my mini. However I used this lot a month ago to grow up some plasmids and it works fine. So I doubt.
3. I kinda rule out religate vectors because 1) there are blunt-stick and non-compatible sticky-sticky ligation (I confess I didn't do dephosphrylation) and 2) If they were religate vectors I should see the vectors in gel, not completely blank.

The next thing I will do is run gel to check 1) my ligation products and 2) my mini elute. I will borrow amp from other groups, as well as mini, and do it again and see.

Do you have any suggestion about my problem?




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