Team:Brown/Notebook/July21

From 2010.igem.org

Wednesday, July 21, 2010

Miniprepped the 8 overnight cultures of pGEM+WillRS ligation blue-white selection colonies (using qiagen kit)

Note - looked at the plate today, it looks suspicious because there are blue colonies, but most are light blue (we chose the ones that were most white.

Double digest of pGEM+WillRS miniprep

20ul double digest with BamHI and Nco1

7.8ul dH2O
2ul 10x NEB buffer 3
8ul DNA (miniprep)
1ul Nco1
1ul BamHI
0.2ul BSA

20ul total volume

Incubated at 37c for 1 hour

Master mix

Made a master mix for 9 rxns:

70.2ul dH2O
18ul NEB buffer 3
1.8ul BSA
9ul Nco1
9ul BamHI

Nanodrop data of WillRS and pGEM (in ng/ul)

Tube 1 - 265.5
Tube 2 - 195.6
Tube 3 - 164.3 (corrected from 235.3)
Tube 4 - 129.8
Tube 5 - 251.5 (corrected from 261.5)
Tube 6 - 171.7
Tube 7 - 142.0
Tube 8 - 208.5

Test of kanamycin

7 Tubes:

Tubes 1-5 are 50ug/ml Kan in 5ml LB with 5ul of XL1s
Tube 6 is 5ul XL1B, no Kan
Tube 7 is non-inoculated 5mL LB

These will be grown overnight

Gel

Cast and ran a 1% agarose gel (for the products of the recent digest). Accidentally ran for a while at 25V, this resulted in a lot of streaking, including the ladder well, so we will have to repeat the digest and gel.

Creation of competent cells

6:00PM - ODed 1mL of cells containing the Lovtap reporter system #2. OD was 0.259@600nm.

6:20 - OD was 0.285

6:48 - OD at 0.300. Placed on ice

6:58 - placed in centrifuge, spun for 5min@3k,4C

7:14 - Saw no pellet. Respun for 10min@5k,4C

7:24 - Saw pellet, added 2ml CaCl2 (forgot to resuspend!). Spun down as above

7:34 - Added 500ul CaCl2 AND RESUSPENDED!

8:08 - Added 1ul of lovtap plasmid to 2 aliquots (250ul each). Lovtap @ 105ng/ul, possibly too high, but these cells are probably not at optimum competence

8:__ - Put on ice, then heat shocked

8:40 - Put in incubator

9:45 - Out of incubator, spread 120ul of cells on 4 LB/Amp/Kan plates