SBB11Ntbk-RishiRawat

Rishi Rawat 12:58, 17 February 2011 (EST)

I messed up the PCR. I used 2.4uL H20 instead of 24uL. MUST REDO PCR.

The CORRECT mixture:
24uL ddH2O
3.3uL 10x Expand Buffer "2"
3.3uL dNTPs (2mM in each)
1uL Oligo 1, 10uM
1uL Oligo 2, 10uM
0.5uL Expand polymerase "1"
0.5uL Template DNA

Made Master Mix(4 reactions):
96 H20
13.2 Buffer 2
13.2 NTP
---> use 30.6uL per pcr tube

Rxns:add to the 30.6uL

1. 1
   

.5 MG1655
1 1118F
1 43R
.5 Pol

1. 2
   

.5 MG1655
1 48F
1 48R
.5 Pol

1. 3
   

.5 MG1655
1 27F
1 27R
.5 Pol

1. 1. 1. not sure how much pol actually got used in this reaction. Perhaps less than 1uL, perhaps a bit more.

Rishi Rawat 02:03, 15 February 2011 (EST)

Recieved oligo RRsb1118Forward, resuspended in water(added 295 uL ddH2O) to make 100uM stock.

Did PCR for sb1118, 1122, 1102 used mg1655 template

sb1118 F: sb1118F R: ss43R goal:371bp

sb1122: F: ss48F R: ss48R goal:1010bp

sb1102: F: ss27F R: ss27R goal:986bp

used extend protocol:

2.4uL h20 3.3uL 10x expand2 3.3uL dNTP 1uL oligo 1uL oligo .5uL expand .5uL Template

Placed reaction tubes in <2k bp PCR Reaction

Rishi Rawat 02:03, 9 February 2011 (EST)

Hello!

Rishi Rawat 02:02, 9 February 2011 (EST)

This is my first entry!