Chris ran all previously digested parts on two 2% gels.
Gel 1
| Ladder |
C0051(U) |
J6504(U) |
J23102(U) |
K145303(U) |
R0010(U) |
K235000(D) |
| |
|
|
|
|
|
|
(U)upstream digest (D)downstream digest
-Layne
Unfortunately this gel was a mess - we didn't bother taking a picture. The bands had trailing edges and all bled into one. The ladder was impossible to interperet. The lack of a clear banding pattern could have been due to the agarose not being completely dissolved before pouring (although I'm pretty sure that it was..)
Gel 2
| Ladder |
B0034(D) |
B0015(D) |
J23066(D) |
E0422(D) |
K235002 (2) (D) |
K235001(D) |
K235002 (1) (D) |
| |
plasmid |
plasmid |
plasmid
part
|
plasmid |
plasmid |
plasmid |
plasmid |
-Layne
This gel was also pretty bad (similar to gel 1). It's not possible to interperet too much from the ladder except to say that it looks like we got bands for the parent plasmid from each digest (similar bands in a row near wells) and the part that was cut out for part J23066 only (single band further down in lane 4).
I've made up some 1.5% gels with a 50% increase in EtBr for future experiments.
To make up two 1.5% gels:
120ml dH2O
1.8g Agarose
24ul EtBr
-the EtBr increase might give us stronger signals.
-The agarose was definitely 100% dissolved this time around, both boiled over.
-Due to boiling over, I only got 3 average thickness gels and one that is very thin. (We're thinking about running this in parallel with another gel with the same DNA in both to compare results.
The ligation products (frozen prior to transformation) of parts k235000, K235001, K235002, K235003, K235004 and K235005 were retransformed into Dh5alpha using the extended incubation time (2 hours) and plated onto Cm plates.
Derek made up a fresh batch of competent Dh5alpha from a seed culture of about OD .3 (as per protocol) and another culture of about OD .4 (they were already at this OD and we want to see what effect this has on competence).
(back to Lab notebook)
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