Lab Aug 11 2009

Miniprep'd I0500 A, I0500 B and P1010 Amp

Made glycerol stocks of I0500 A, I0500 B, P1010 Amp, ccdB strain

Made more competent ccdB resistant cells (301)

Transformed R1051 (pcI)(amp), I0500 (pAra)(kan), J23102 (const. p)(amp)(plate 1, 18G) and E0422 (ECFP tripart)(amp)(plate 1, 8M)

Poured Cm plates

Started broth cultures of DH5alpha, P0151 and K145303 (sketchy results).

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Layne 9am

We now know that both of the parts we plated on Kan were supposed to be Amp, so it's not surprising we weren't successful with those. (R0010, R0051)

Three of the five plates from yesterday showed growth:

P0151 - Lots of colonies

K145303 - 2 colonies

R0051 - 2 tiny colonies.....

R0010 - nothing

R1051 - nothing

Recent transformation parts list:

P0151 - tripart (RBS + lambda cI inhibitor protein + stop). on plasmid pSB1A2, kit plate 1 well 10C

K145303 - quadpart (promoter + Ribolocked RBS + GFP + stop). on plasmid pSB1A2, kit plate 2 well 6H

R0051 - lambda cI regulated promoter. on plasmid pSB1A2, kit plate 1 well 6K

R0010 - misslabeled

R1051 - lambda cI regulated promoter. on plasmid pSB1A2, kit plate 1 well 8E

-Subcultured ccdB resistant strain in preparation for making cells competent. Inoculated 2.5ml from overnight broth culture into 250ml LB and put into shaker at 37 degrees. Waiting on OD .3 now.

-Made glycerol socks of I0500 A, I0500 B, ccdB resistant strain and P1010 Amp backbone.


 * All cultures were at stationary phase from overnight while the protocol suggest log phase is best.

- .6ml of 40% Glycerol and .6ml of broth culture. Stored in -80 later on (**Should be stored in -80 right away)

-Miniprep'd both I0500 broth cultures and P1010 Amp backbone plasmid, stored plasmids in -20

-Poured Cm plates

-Made 6 aliquots of competent ccdB using the 301 competence protocol:

-Used 10X the amounts described in protocols. ie 200ml broth culture, 50ml CM1, 6ml CM2.

We have decided to switch back to the Top10 competent cells procedure.

We have decided to start with R0010 and J23066 for our first system. These are a lacI promoter and a ribokey (including terminator). These are parts that are successfully frozen in the -80oC. They both have Amp resistance.

1.) Since our ccdB resistant cultures with Kan resistance and Chloramphenicol resistance failed, we have to try again.

2.) We are out of competent DB3.1 cell stocks so our first step is to grow more of these

3.) Then make them competent.

4.) Transform with ccdB from Kan and ccdB from Chl

Transform and plate today:

R1051 (pcI)(amp)

I0500 (pAra)(kan)

J23102 (const. p)(amp)(plate 1, 18G)

E0422 (ECFP tripart)(amp)(plate 1, 8M)

Also spun down, resuspended, and plated last of K145303 (ribolocked GFP)(amp)


 * We decided to continue on with the 301 protocols for making cells competent that we had already started this morning. The reasons being: the only argument we had going for the Top10 protocol was to stay consistent with majority of our transformations, the big centrifuge tubes would not be accessible that early in the morning, these cells will only be useful for transforming in P1010 backbone plasmids (need two more), the 301 protocol had already been started that day and is now completed.  If we decide we do need these cells made competent the Top10 way, we can potentially throw on a batch and start more Dh5alpha at the same time

Tomorrow:

1. Run both I0500s on a gel with ladder (could throw P1010 on too?) ((We'll want to run K145303 as well, so depending on when it's miniprep'd))

2. Transform P1010 Kan and Cm backbones into competent ccdB (with pUC as control)

3. Make glycerol stocks of Dh5alpha, P0151, K145303

4. Miniprep P0151 and K145303 (if they're not needed right away we could miniprep all 6 on Thurs?)

5. Start broth cultures of todays successfull transformations

6..... Forgetting anything?

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