Gibson assembly continued #

Transformation results #

Picked up transformed colonies from yesterday’s Gibson assembly reactions. Results were not great. Negative control had about 5 colonies and only plates that were streaked had any visible colonies. On average these plates had about 5x the number of colonies as the negative control giving me some hope there might be successful transformations.

For transformations that failed I replated with the transformed cells from yesterday which were kept at 4C using 100 ul of the transformed cells and spreading via beads. I put these plates in the 37C room at around 10:30. For plates with colonies I (VR-25, 17, 18, 28, 20, 21, 22 and 26) harvested 5 individual colonies from each plate and began LB cultures of each colony at 37C with shaking in the hot room.

The platinum loop is broken so I hade to dig out colonies with more of a platinum stick resulting in very large chunks of agar coming with the colonies.

Finished LiCL purification of

9/4/1 insert mini preps #

Finished LiCl purification of Gibson samples that I started yesterday. Nanodrop results for all samples are below and is also available at this spreadsheet in sheet 9-7-21.

Insert	Colony	Yield	Total DNA (ug)	Additional ID
1	1	102	51
1	2	97	48.5
1	3	166	83
1	4	131	65.5
1	6	167	83.5
1	7	219	109.5
2	2	261	130.5
2	3	193	96.5
2	4	137	68.5
2	5	162	81
2	6	276	138
2	7	136	68
4	1	198	99
4	2	157	78.5
4	2	192	96
4	5	216	108
5	1	273	136.5
5	2	177	88.5
10	1	160	80
10	2	501	250.5
11	3	372	186
11	4	232	116
11	5	144	72	B
11	5	203	101.5	A
11	6	199	99.5
14	1	216	108
14	2	317	158.5
14	3	224	112
14	4	123	61.5	B
14	4	411	205.5	A
14	6	326	163
14	7	195	97.5

Overall preps look good DNA wise. I then ran samples out on a gel (TAE 120V 1hr).

There seems to be type groups of bands, one running slightly higher than the other. This looked to me like how PCR products from vectors are longer than those from fragments and I checked again to see if any inserts were actually from vectors and not fragments; VR-4 is. But I only prepped 7 samples of VR-4 and there are more high bands than that. Sending samples to sequencing tomorrow so we will see how that goes. Samples are already prepared and are stored in the kitchen freezer. Just need to be dropped of at LSA before 5 tomorrow.