Tom Jerry

Hello,

I am planning on ordering a new comb for my GeneMate "Mini-Plus Horizontal Gel System (9x11cm)", one which will provide more loading volume than my current 10-well, 1.5mm comb.
The problem is that I couldn't find an 8-well, 1.5mm thick comb which I looked forward to buying.

Here is the catalog:
http://internal.bioe...600615000615065
Only an 8-well, 1.0mm comb is for sale, which I believe holds the same volume as the 10-well 1.5mm which I already have.

Currently I am looking for an alternative comb to purchase.

1. Can somebody please tell me what a "9-well comb, multichannel compatible, 1.5mm thickness" is? Will this comb hold more volume than my 10-well, 1.5mm comb? Or is it the same-sized wells, just with more space in between each well? I don't want to risk buying these because each cost ~$50.

2. Also, because the "Midi" Horizontal Gel System (12x14cm) has an 8-well, 1.5mm comb available on the catalog, I was wondering if that would fit into my "Mini-Plus" casting tray if I decide to get it. I understand that my "Mini-Plus" width is 9cm and that the "Midi" width is 12cm, but can I still buy a "Midi" comb and use it for my "Mini-Plus"casting tray?

3. My last choice is to go with the "Mini-Plus" 5-well, 1.0mm comb. This, definitely will hold more than enough volume -- it's just that sometimes I have more than 5 samples to load at once.

Does anybody have an idea which comb I should buy? I am open to any suggestions. Thank you for your support!!

I'm still new in my research lab, and I've realized that a lot of the plasmids that I use are in N1 or C1. (e.g. pRSETa in N1, PAmCherry1 in C1) When my PI says they're "mammalian," does she mean that these circular plasmids readily exist in human cells? If so, how/why do we use them? If not, how and from where have these N1 and C1 been isolated? Also, do the N and the C have to do anything with the amino and carboxyl termini of amino acid chains?

I really need some help.
Thank you!

Hello,

For some reason, my digestions are not working. I am afraid to consult my PI because the cause of the problem might be something that has to do with my procedures rather the actual supplies that I am using. I hope someone can pinpoint what I am doing wrong. Thank you!

Experiment goal:
1. Create insert with sticky ends by introducing restriction sites (Kpnl and Xbal) through PCR, then digesting those ends (with Kpnl and Xbal) after PCR product purification. This should create one band on the gel.
2. Also digest a vector plasmid with the same two enzymes. Since the plasmid is circular this should create two bands, a bigger fragment (which I will extract to use as the vector) and a smaller fragment to toss.
3. Gel extraction, ligation of vector and insert, then transformation.

After running the digestion products of 1 and 2, I saw two bands (instead of one) in the PCR product and only one band in the plasmid lane (instead of two). After making this observation I repeated the entire PCR to start all over, only to visualize the exact same results on the gel even on my second try. This made me want to test the Restriction Enzymes, so here's what I did.

I obtained three different plasmids that each had one Kpnl and one Xbal site, and prepared their digestions in the following order:
1. MB grade water (18.5uL)
2. 10X Thermo Scientific Fast Digest Buffer (3 uL)
3. 2.0 ug vector (= 2.0 uL since the concentrations of all the plasmids were 1ug/uL)
4. Fast Digest Kpnl (2.5 uL)
5. Fast Digest Xbal (2.5 uL)
6. Fast AP Theremosensitive Alkaline Phosphatase* (1.5 uL)
Total volume = 30 uL
Mixed lightly, centrifuged quickly, then incubated at 37C for 1 hour.

*Here I decided to use the Alkaline Phosphatase because I was planning to use the digested fragments for ligation later on if they worked.

However, electrophoresis showed only one fragment for each plasmid lane, all at their original lengths (~4kb).

Because none of them worked, I then decided to repeat the same procedures but now with a new stock of Kpnl and Xbal. I actually prepared three tubes for the same Kpnl and Xbal that I used above, and three other tubes for the new set of enzymes. In total, I ran 6 lanes on the gel. I also altered the incubation duration to 15 min because I realized that I was using reagents for "Fast Digest". However, visualization showed no digestion; I only saw six big bands near the top of the gel, each at their own original fragment size.

Hmm... if it's not the enzymes that aren't working, what could be wrong? I also think the plasmids are in great condition because it's very hard to believe that all three of them aren't in good shape. Plus they have been working when my other lab members used them.

Thank you very much for your time!

~Tom