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zhgljj1998's Content

There have been 21 items by zhgljj1998 (Search limited from 20-January 19)


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#11514 expression of small peptide

Posted by zhgljj1998 on 28 February 2005 - 08:29 AM in Protein and Proteomics

design another peptide with Tyr, other AA are same. then you got idea to get concentration of your small peptide.



#11245 PMSF

Posted by zhgljj1998 on 21 February 2005 - 05:08 PM in Molecular Biology

protease inhibitor



#10993 BamH1 and Xho1 double digestion problem

Posted by zhgljj1998 on 13 February 2005 - 12:48 PM in Molecular Biology

You won't see size difference between the single-digested or double-digested, as you said, but you will be able to see if both enzymes are able to cut the vector in the conditions you use for the double digestion. If the BamHI buffer is not optimal for XhoI digestion, then maybe in the 4h incubation, XhoI is not able to digest, so in the control single digestion with XhoI using BamHI buffer you will see lots of supercoiled DNA and just a bit of linearized plasmid- this will mean that most of your plasmid in the double-digest has been in fact digested only with BamHI. Then maybe you need to add more XhoI, increase the incubation time, or do sequential digestions. Hope it helps!

Thank you very much, Dr Badcell :wacko: , try what you suggested. finally, finally, get right sequence result back. my problem is fixed. anyway, i digested one by one, and incubate more than 10 hr in 37 degree water bath either. :D



#10992 Tricine SDS PAGE

Posted by zhgljj1998 on 13 February 2005 - 12:39 PM in Molecular Biology

Did anybody try Tricine SDS gel to see small peptide about 1.5 KD before? I searched some protocol by google how to make the tricine gel, but the resolution is not good. can somebody tell me any tips or your method to make the gel? thank you.



#10621 Where are you from?

Posted by zhgljj1998 on 01 February 2005 - 03:07 PM in Chit Chat

Hi,

CAn you only tell me where do you from?

Sebela (Quebec, Canada)
;)

Beijing, China, now in London.



#10556 cloning from PCR

Posted by zhgljj1998 on 31 January 2005 - 07:19 AM in Molecular Biology

i'm PCRing my gene (1.1kb) out from a plasmid template to create different restriction enzyme sites at the ends.  I gel purify the PCR product using a kit from Qiagen and elute with 10mM TrisCl, pH8.0.  I get a good recovery....i  check this by running it out on a gel. from this estimated conc. i digest with BglII and XbaI and gel purify and test conc. the same as above.  from this i set up a ligation in a vector:insert  ratio of 3:1, 1:1, and 1:3 with ligase from Fisher.  i transform all 10ul of ligation reaction into 100ul of competent cells and plate 100ul of the transformed cells, but still get no colonies.

what seems strange is after digesting, the vector (~3.2kb) shows a clear exised band of about 40bp, the fragment between BglII and XbaI, whereas my digested PCR product shows no small band, even when I ran a 20% acrylamide gel to look for extra small fragments (5bp and 8bp from each primers' 5' end).  is this something i should be worried about?  how can the enzyme sites NOT be there when I get a very dense band from my PCR reaction at 1.1kb?

man, how can you see the bank about 40 bp, 4%argrose gel?, i am going to check my double digested dna between BamH1 and xho1 (40bp) now, so need your suggestion, thank you.



#10462 Histag, N or C terminal?

Posted by zhgljj1998 on 28 January 2005 - 07:48 AM in Molecular Biology

is there any big effect for the HisTag to be designed in N terminal and C terminal? which one is better for purify protein? i am fist year in research, need suggestion, thank you.



#10446 Reuse TAE buffer and agarose gel?

Posted by zhgljj1998 on 28 January 2005 - 02:55 AM in Molecular Biology

Hi all,

I've just started in a new lab, and here they re-use their 1X TAE for DNA gels up to 5 times. I was under the impression that it should only be used once, however  TBE could be used around 3 times.

So, should you, or should you not re-use TAE?

Thanks alot....

Ros[B][COLOR=red]

if you do not cut your gel, then reuse it until someone else change the buffer. :lol:



#10409 BamH1 and Xho1 double digestion problem

Posted by zhgljj1998 on 27 January 2005 - 10:06 AM in Molecular Biology

Have you done separate digestions with both REs to see if both of them are working in the conditions you use? I usually do scaled-down versions of my double digestion (10 microliters instead of 50, f.i.) but using only one of the REs, for the same time and with the same buffer, as a control. That's especially useful when the buffer is not the optimal one for one of the enzymes.

i am trying digestion one by one at the moment. (BamH1 first, then, Xho1). i am going to try total 10 microliter volume of double digestion tomorrow as you suggested. but i am not sure the control is quite useful since there are only 40 bp between BamH1 and Xho1. i may not see any difference between double digested sample and control. do i really need to run 4% gel to see the 40bp, then make sure i get double digested vector? really thank you, man.



#10400 Dephosphorylation of vector before ligation

Posted by zhgljj1998 on 27 January 2005 - 08:03 AM in Molecular Biology

Yeah, that dirty little trick has spared me a lot of minipreps... :)

Digest the Ligation reaction. man, you are good. by the way, about the Miniprep, actually, how much DNA can you get from one miniprep? sometimes, i got very low concentration. is there any trick? thanks.



#10392 BamH1 and Xho1 double digestion problem

Posted by zhgljj1998 on 27 January 2005 - 04:52 AM in Molecular Biology

Did anyone use BamH1 and Xho1 double digestion?

i used pet28a as DNA 36 (micro l)
BamH1 2
Xho1 2
10xBSA 5
10x BamH1 Buffer 5
TOTAL 50 waterbath 37 degree 4 hour, gel extraction

T4ligase 4 degree 2 O/N, found self-ligation?

can anyone help me?



#10079 Colony PCR for clone screen

Posted by zhgljj1998 on 18 January 2005 - 02:57 AM in Molecular Biology

Hi, is there anybody using Colony PCR to check your plasmid before sending sequence. is it necessary? thank you.



#10038 Problem in the Digestion

Posted by zhgljj1998 on 16 January 2005 - 10:26 AM in Molecular Biology

i double digest a similar vector with BamH1 and EcoR1, with no problems.  I put both enzymes in together with y*/Tango buffer and leave it at 37'C for ~2-3 hours.  Works fine. 
how long is your digestion?
are you putting in enough enzyme?
can't think of anything else that could go wrong.
Try cutting with some other enzymes to compare.

hi, man, thanks for your suggestion. I tried one more week, then, i quit to double digest with BamH1 and EcoR1 in the vector which is next to each other. just i am going to change other construct. i thought your double digest works well is amazing and lucky maybe. is there any special in your protocol?



#10026 Difference between Luria Agar and Luria-Bertani

Posted by zhgljj1998 on 15 January 2005 - 07:14 AM in Molecular Biology

Luria Agar or Miller's LB Agar contains high salt  (10g NaCl/L) while LB (Luria-Bertani) contains 5g NaCl.

For transformation with DH5a, which one is better? thanks



#9994 Difference between Luria Agar and Luria-Bertani

Posted by zhgljj1998 on 13 January 2005 - 11:40 AM in Molecular Biology

Is there anybody know the difference between Luria Agar and Luria-Bertani? I will make plates with Kanamycin, which one is better?any suggestion?



#9993 ligation question

Posted by zhgljj1998 on 13 January 2005 - 11:36 AM in Molecular Biology

Do you mean to run a gel using the ligation reaction? It won't hurt if you do so and may give you some idea whether your ligation is successful or not.

yes, can we exactly make sure the ligation work well or not from gel with control? thanks,man.



#9984 BamH1 and Nde1 double digestion problem

Posted by zhgljj1998 on 13 January 2005 - 08:47 AM in Molecular Biology

HI,
 
  I am using BamH1 and Nde1 to double digest the PCR product and Vector.The BamH1 and Nde1 are from NEB. I try using NEBuffer2 as buffer to digest,but the cleavege is poor.
Anyone has ever used these two REs together for double digestion? Which buffer do you use? Any tricks to increase the the efficency?


Thanks!

netnus

do not use buffer2 please. i had been troubled for two month. try to use other buffer. good luck



#9981 ligation question

Posted by zhgljj1998 on 13 January 2005 - 06:48 AM in Molecular Biology

can anybody tell me, is it useful to run the ligation mixin the gel?really confused since somebody suggested to do this step. Also, the Insert : Vector (mol)=? be better, 1:1; 1:3; 3:1????????or depend on your stuff?thanks.



#9969 Problem in the Digestion

Posted by zhgljj1998 on 13 January 2005 - 03:19 AM in Molecular Biology

i double digest a similar vector with BamH1 and EcoR1, with no problems.  I put both enzymes in together with y*/Tango buffer and leave it at 37'C for ~2-3 hours.  Works fine. 
how long is your digestion?
are you putting in enough enzyme?
can't think of anything else that could go wrong.
Try cutting with some other enzymes to compare.

Thanks. I tried to digest 2.5; 4 and 5 hr. Just the Enzyme and buffer i used were NEBuffer (Biolab). never used Y+Tango. `tried 1 and 2 microlitre Enzyme. no self ligation as well. Maybe ther is to much insert (3:1)?it made me mad for 2 months. I thank you anyway.



#9937 Problem in the Digestion

Posted by zhgljj1998 on 12 January 2005 - 10:57 AM in Molecular Biology

I used BamH1 and EcoR1 to cut pET-28a (5.5kbp), BamH1 is next to the EcoR1 in the plasmid. I tried to Double Digest and digest one by one. after ligation, no transformation colony. After checking everything, I thought it must be the problem of Digestion. is it possible that the plasmid like this (BamH1 Next to EcoR1) can be cut very well since we cannot see any differrence between double-cut and single-cut.

If we digest the vector by En one by one, Ecor1 first or BamH1 first, and need change buffer after the first En digestion? Please Help.



#9915 ligation again

Posted by zhgljj1998 on 11 January 2005 - 02:28 PM in Molecular Biology

hi, did you fix your problem by now? i think the problem is Vector digestion. your vector only digested by BamH1 or EcoR1, then, all the vector ligased with the insert, that can explain why you cannot get anything from transformation. if you fix the problem, please let me know, i was nearly mad for the same problem




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