Gibson assembly and big IVT attempt #

Gibson assembly #

Prepared three Gibson assembly reactions detailed on table 10/5/21 of the Gibson assembly spreadsheet using samples pFC9-9-10-21 sample 1 as the backbone vector. Diluted all Vr inserts to 50 ng/ul before adding to reaction mix. At 9:30 AM I placed Gibson assembly reactions on ice to help Tadas and decided to only move forward with samples VR 8, 12 and 15 plus a negative control to check the reagents were still good before expending a large number of chemically competent cells.

After incubating samples for 1 hours at 50C followed NEB electro-competent E. coli transformation protocol and plated the resulting transformed cells onto Amp agar plates. Incubated plates at 37C in hot room overnight.

IVT reactions #

Wanted to set up a large IVT gel to potentially show to math collabortors for my presentation to them tomorrow. However, we were out of NEB RnaseH1 and so had to use lab stock RnaseH2 whose activity was in question. Other than this change I followed the lab IVT protocol with samples VR 1, 2, 3, 4A, 5A, 10, 14, 13, 18, 20, 28, 29A and pFC9.

IVT gel #

Ran IVT reactions on 0.8% agarose 1x TBE gel (no EtBr) in 1x TBE for 2 hours at 60V. Lane layout is below. Each sample has three lanes first is the untranscribed control, second is transcribed RNAseH2 treated and final is the transcribed untreated lane.

Lane	Sample	Treatment	Lane	Sample	Treatment
1	VR-1	None	20	VR-14	T7 + RnaseH2
2	VR-1	T7 + RnaseH2	21	VR-14	T7
3	VR-1	T7	22	VR-13	None
4	VR-2	None	23	VR-13	T7 + RnaseH2
5	VR-2	T7 + RnaseH2	24	VR-13	T7
6	VR-2	T7	25	VR-18	None
7	VR-3	None	26	VR-18	T7 + RnaseH2
8	VR-3	T7 + RnaseH2	27	VR-18	T7
9	VR-3	T7	28	VR-20	None
10	VR-4A	None	29	VR-20	T7 + RnaseH2
11	VR-4A	T7 + RnaseH2	30	VR-20	T7
12	VR-4A	T7	31	VR-28	None
13	VR-5A	None	32	VR-28	T7 + RnaseH2
14	VR-5A	T7 + RnaseH2	33	VR-28	T7
15	VR-5A	T7	34	VR-29A	None
16	VR-10	None	35	VR-29A	T7 + RnaseH2
17	VR-10	T7 + RnaseH2	36	VR-29A	T7
18	VR-10	T7	37	pFC9	None
19	VR-14	None	38	pFC9	T7 + RnaseH2
			39	pFC9	T7

Unfortunately, gel is not clean enough to show to math people but does show variability between plasmids to some degree. However, it is difficult to determine how effective RnaseH2 treatment was as resolving R-loops.