Miniprep and electro-competent cell transformation results #

Minipreps #

Came in early to miniprep samples VR 12, 15, 16, 25, 23 and 21 that I grew overnight cultures of yesterday. I followed my miniprep protocol and then ODed the samples after LiCl precipitation of DNA the results of which are shown in the table below and are also recorded in table 10-8-21 of nanodrop spreadsheet.

Yields #

Insert	Colony	ng_per_ul	Volume at measurement	Diluted Volume	Final Concentration
12	1	1461	40	80	730.5
12	2	1485	40	80	742.5
12	3	1296	40	80	648
12	4	1296	40	80	648
12	6	1293	40	80	646.5
15	1	1253	40	80	626.5
15	2	1938	80	80	1938
15	3	1480	40	80	740
16	1	1368	40	80	684
21	1	1262	40	80	631
23	1	1408	40	80	704
23	2	1436	40	80	718
23	3	1456	40	80	728
23	4	1323	40	80	661.5
23	5	1412	40	80	706
25	1	1366	40	80	683
25	2	1275	40	80	637.5
25	3	1401	40	80	700.5

Yields looks good, digestion and sequencing will reveal if preps actually contain successful insertions.

Digestion with KpnI #

I digested 600 ng all prepped samples with 0.5 ul KpnI-HF in 25 ul reaction volumes for 1 hour at 37C. If a VR insert is present in the plasmid this will introduce an additional KpnI site compared to pFC9 alone and produce 2 bands instead of 1. Stella loaded this gel.

0.8% agarose
1x TAE buffer and gel
0.04 ul / ml EtBr in buffer and gel
120V for 40 minutes

Lane	Sample	Lane	Sample	Lane	Sample
2	12-1	8	15-1	15	23-4
3	12-2	9	15-2	16	23-5
4	12-3	10	16-1	17	25-1
5	12-4	11	21-1	18	25-2
6	12-5	12	23-1	19	25-3
7	12-6	13	23-2
8	15-1	14	23-3

Lane 1 is a 1kb ladder.

Appears that most samples were cloned successfully (with the insert) except for 23-1 and 23-4.

Transformation of electro-competent cells #

Picked up transformed electro-competent cells I plated yesterday and was again disappointed by relative lack of cell growth, although there was some. Colony counts are shown in the table below.

Sample	Plated volume	Colonies
C	150	1
C	250	1
31	250	0
31	150	0
20	250	2
20	150	0
17	250	1
17	150	2
27	250	1
27	150	3

It was also concerning to see that both negative controls (only transformed with pFC9 fragment) had a single colony. There are not that many colonies so I am going to go ahead with preps anyway and hope for the best with these bugs.