SBB11Ntbk-RishiRawat

Rishi Rawat 14:04, 24 February 2011 (EST)

Goal: do Zymo gel extraction cleanup for the solubulized gels from 2-22-10, corresponding to SBB1102, 1118, 1122

Accessed solubulized gel samples

Added to Zymo Column + Spin

[ + 200uL A4(Wash Buffer) + spin](x2)

spin 90sec

elute 9uL ddH20

Samples are ~called [RR1102, RR1119, RR1122] RE Digest, Zymo, Insert

Next Step: Ligation with Vector(~1hr)

and Transformation by Heat Shock(~10min + 1hr recovery)

and Plating(~30min)

Rishi Rawat 13:50, 22 February 2011 (EST)

CA redid all of the PCRs(for gels see post: "JCAnderson 19:32, 21 February 2011 (EST)" at http://openwetware.org/wiki/SBB11_gels).

Then he did PCR cleanups for each rxn.

I am starting the day with SB1118, SB1122, SB1102.

goal: do digestions.

Protocol: 8ul pcr product, 1 uL NEB2, .5uL EcoRI, .5uL BamHI

-->Mixed materials

-->Ran Prep. Gel(http://openwetware.org/wiki/Image:022211-PrepGel4.jpg) with complete digestion mixture(10ul) + 1uL loading Dye.

---The last three lanes correspond to 1102, 1118, 1122(in order from L to R)

---the main bands of all three samples are approx. the right size

---1118 did not work as well as 1102 or 1122.

---1102 displays multiple bands.

--> extracted the main band for each sample

--> added 600uL buffer(yellow), in brown tube, to bring gel into solution; placed at 55C for approx 7 min + inversions

--> stored in box, probably to be kept at -20C - 4C

Rishi Rawat 16:29, 18 February 2011 (EST)

Ran analytical gel:

Lanes 6,7,8 represent PCRs 2, 1, 3. All three PCRs Worked. but pcr 1 was weak.

http://openwetware.org/wiki/Image:021811-AnalGel1.jpg I expected 1kb, ~380 bp, 1kb. The band at ~380 was too faint.

I need to redo the PCR: cycle 2k45 with + DMSO(number 5) and -DMSO(number4)

---

I also did pcr cleanups for 1,2,3:

PCR cleanup:
180 ADB
Zymo Column -> spin
200ul Zymo Wash(x2)
90 sec spin
elute 30ul

These pcr products are stored.

Rishi Rawat 14:05, 17 February 2011 (EST)

NEXT STEPS:

Day1:
1.) Analytical gel
Run: 2(~1kb), 1(~300bp), 3(~1kb)

2.) (20Min) PCR cleanup; Regular Zymo Cleanup(frag>200bp) http://openwetware.org/wiki/Template:SBB-Protocols_Zymo1; elute in 33uL

3.) (1hr) RE Digest Eco/Bam; http://openwetware.org/wiki/Template:SBB-Protocols_Enz2
4.) Run Preparative Gel

Day2:
4.5) (30min) Gel purify; http://openwetware.org/wiki/Template:SBB-Protocols_Zymo3

5.) (30min)Ligate; http://openwetware.org/wiki/Template:SBB-Protocols_Enz4

6.) (1hr)transform; http://openwetware.org/wiki/Template:SBB-Protocols_Micro1

7.) colony stuff...

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

2.)
.5 MG1655
1 48F
1 48R
.5 Pol

3.)
.5 MG1655
1 27F
1 27R
.5 Pol

• ////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!