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From 2010.igem.org

8/31/10

Results of transformation - the plates had growth but (no) distinct colony pattern. The amp that was added to the Agar seemed to have been destroyed before plates were poured. The Agar could have been still too hot and killed the Amp. So today, we will make fresh LB/Amp Agar and pour new plates.

1) Grabbed the Powder LB Broth Lennox we weighed 5.002 g LB and added this to a Capped 500 mL Elenmiyer flask then added 250 mL 18 ohm DI water then shook to mix then placed into autoclave on liquid setting for 15 min at 121 C

2) We will let the LB cool then add

5.002 Difco tm LB Broth Lennox Ref# 240230 500 g container Lot# 9082989 Ex. Date 2014/02/28 Date received 6/8/09 G.M. opened 4/9/10

Autoclave – Agar, molecular Biology grade CAT# A7777 Lot# A777715E0901 Re Assay Date 5/15/14

To test our

Amp we will make another 250 mL LB Agur and add Amp to it in different doses using a sterile tube. We will be making 4 plates at different concentrations of Amp So 15 mL with no Amp 15 mL with 15 mL Amp 15 mL with 30 mL Amp and 15 mL with 60 mL Amp then pour into plates. We forgot to add agur to the LB Broth so I added 3.752 g to 250 mL LB Broth into 2 500 mL capped Elenmiyer flasks 250 mL in each 3.75 g Agur in each now heating on 2 hot plates. Then we will add Amp to plates described above. removed

8/31/10

After agar is dissolved, and allowed to cool, I will add Amp in different doses to specific plates.

1) I decided to use microwave to dissolve the Agar into the LB/Broth using the 4oz beverage setting I ran intermittently not allowing to boil over.

2) After dissolved the autoclaved 250 mL flask and put the other into a 40 C hot water bath to cool rapidly this one I will use for my 4 plates.

3) After cooling in water bath I removed lid on Elenmyer flask and checked temp it was 41 C then set up in Biofume Hood added 15 mL Agar with Amp and poured four plates. 1 – No Amp 2 – 15 mL Amp 3 – 30 mL Alp 4 – 60 mL Amp 1 & 2 the lids were opened in the environment for a sec.

4) I placed our 4 plates into a plate bag and put into fridge in the back room. 5) We will plate next time. 8/31/10 J.H.

--Rchamberlin 18:34, 30 September 2010 (UTC)