"

From 2010.igem.org

Lab work

Emulsifier

The transformations of yesterday containing the different ligation reactions gave colonies. All plates contained red and white colonies. This is probably due to the fact that we did not purified the backbone before ligation. So some plasmids reassembled. Pieter picked 5 colonies of every plate (no red ones) and 1 from the control plate (a red colony) and performed a Colony PCR to check which colonies contained the right insert. At the same time he cultivated the colonies in 5 mL LB medium containing the 15 μg/mL name Tetracycline to grow over night at 37 °C.

The PCR products were run on gel a 1% agarose gel.

Lane Description

#	Description	Expected lenght (bp)
0	SmartLadder
1	Transformants 1 of Ligation mix : L2 sample 1 (10 μL + 2 μL loadingbuffer)
2	L2 sample 2 (10 μL + 2 μL loadingbuffer)
3	L2 sample 3 (10 μL + 2 μL loadingbuffer)
4	L2 sample 4 (10 μL + 2 μL loadingbuffer)
5	L2 sample 5 (10 μL + 2 μL loadingbuffer)
6	L3 sample 1 (10 μL + 2 μL loadingbuffer)
7	L3 sample 2 (10 μL + 2 μL loadingbuffer)
8	L3 sample 3 (10 μL + 2 μL loadingbuffer)
9	L3 sample 4 (10 μL + 2 μL loadingbuffer)
10	L3 sample 5 (10 μL + 2 μL loadingbuffer)
11	L4 sample 1 (10 μL + 2 μL loadingbuffer)
12	L4 sample 2 (10 μL + 2 μL loadingbuffer)
13	L4 sample 3 (10 μL + 2 μL loadingbuffer)
14	L4 sample 4 (10 μL + 2 μL loadingbuffer)
15	L4 sample 5 (10 μL + 2 μL loadingbuffer)
16	L1 sample 1 (10 μL + 2 μL loadingbuffer)

L2 Expected length of R0011 + B0032 = 55 + 13 = 83 bp (without flanking primer regions). On gel there are only bands poorly visible at height of 1500 bp. So the transformation failed.

L3 Expected lenght of OprG + B0015 = 744 + 130 = 870 bp (without flanking primer regions). On gel bands are visible of about 1000 bp in lanes 6,7 and 10. The bands just a little lower are unidentified.

L4 Expected length of AlnA + B0015 = 1107 + 130 = 1237 bp (without flanking primer regions). On gel bands are visible of about 1400 bp in lanes 11,13 and 15.

L1 Expected length of J04450 is 1090 bp (without flanking primer regions). On gel one band is visible in lane 16 at height 1400 bp.

From this gel it is clear that the transformation of the Promotor and RBS has failed. The OprG with stopcodon is probably in cultures 6 and 7. Since bands of AlnA with stopcodon and J04450 are about the same size, it is not clear whether this transformation succeeded. Tomorrow I will check the isolated plasmids on gel.