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      <page pageid="24657" ns="0" title="RecA: July 10 - September 28">
        <revisions>
          <rev xml:space="preserve">&lt;html&gt;
7/9 Saturday&lt;/br&gt;
•	added DPN1 to RecA1 mutegensis  &lt;/br&gt;
•	incubated 4-6 hours &lt;/br&gt;
•	PCR purfified  &lt;/br&gt;
•	transfromed RecA1: &lt;/br&gt;
o	A3: RecA1 III from 7/7 T.C.: 5.0 &lt;/br&gt;
o	A5 : RecA1 V from 7/7 T.C.:5.2 &lt;/br&gt;
o	B3: : RecA1 III from 7/8 T.C: 5.0 &lt;/br&gt;
o	B5: : RecA1 V from 7/8 T.C. 5.0 &lt;/br&gt;
•	Transfomred test: T.C.: 2.2 &lt;/br&gt;
•	plated transformations and made freezer stock of RecA1III &lt;/br&gt;
 &lt;/br&gt; &lt;/br&gt;

7/10 Sunday  &lt;/br&gt;
•	got sequencing results back of RecA1 III and V. III is good even when blasting with NCBI,  &lt;/br&gt;
•	V is definetly reversed &lt;/br&gt;
•	test did not grow &lt;/br&gt;
•	ran gel of its PCR purification think had to much lyse because came out smeared just below 1000bp &lt;/br&gt;
•	began digest of PCR purfication of part 2 from 7/8 miniprep of part 1 from ⅞ &lt;/br&gt;
•	litgate &lt;/br&gt;
•	transformed &lt;/br&gt;
•	plate &lt;/br&gt;
 &lt;/br&gt; &lt;/br&gt;
7/11Monday  &lt;/br&gt;
•	centrifuge broke &lt;/br&gt;
•	nothing grew of test plates &lt;/br&gt;
 &lt;/br&gt; &lt;/br&gt;
7/12 Tuesday  &lt;/br&gt;
•	centrifuge fixed. Anisha miniprep RecaA1 &lt;/br&gt;
•	grew Part 1 and Part 2 from registry &lt;/br&gt;
 &lt;/br&gt; &lt;/br&gt;
7/13 Wednesday  &lt;/br&gt;
•	part cultures did not grow because I forgot to transform &lt;/br&gt;
•	transformed from registry &lt;/br&gt;
o	part 1 T.C.: 2.8 &lt;/br&gt;
o	Part 2 T.C.: 2.6 &lt;/br&gt;
o	cultured &lt;/br&gt;
•	RecA1 mute results back--&gt; mutation didn’t work  &lt;/br&gt;
•	New mutegeneiss PCR with extention time 2min 15 seconds to go overnight &lt;/br&gt;

 &lt;/br&gt; &lt;/br&gt;
7/14Thursday &lt;/br&gt; 
•	added DPN1 to RecA1 mute PCR, left to incubate &lt;/br&gt;
•	transformed &lt;/br&gt;
o	T.C.: 5.0 &lt;/br&gt;
•	plated &lt;/br&gt;
•	mini prep part 1 and part 2 &lt;/br&gt;
•	PCR insert Part 2 &lt;/br&gt;
•	digested &lt;/br&gt;
•	ligated &lt;/br&gt;
•	transformed: T.C.: 2.2 &lt;/br&gt;
•	Byron started another PCR of RecA1 mute to run over night &lt;/br&gt;
 &lt;/br&gt; &lt;/br&gt;

7/15Friday  &lt;/br&gt;
•	colonies grew of test and RecA1 but very small so will let grow one more night and PCR colony the next day &lt;/br&gt;
•	Byron &lt;/br&gt;
o	added DPN1 and incubated for 6 hours &lt;/br&gt;
o	heat killed at 80 degree fro 20 min (this should not have happened and probably why mutegensisi didn’t work) &lt;/br&gt;
o	PCR purified product &lt;/br&gt;
o	transformed &lt;/br&gt;
	T.C.: 2.0 &lt;/br&gt;
o	plated &lt;/br&gt;
 &lt;/br&gt; &lt;/br&gt;
7/16 Saturday &lt;/br&gt; 
•	colony PCR test: 2 from each plate 1A, 1B, 2A, 2B, 3A, 3B    nothing grew on plate 4 &lt;/br&gt;
•	made culture of RecA1 mute 3 from one plate : 1A, 1B, 1C. Other plate only had one colony &lt;/br&gt;
•	forgot to add oil before PCRing, started another PCR whil cultureing all sample &lt;/br&gt;
 &lt;/br&gt; &lt;/br&gt;

7/18  &lt;/br&gt;
•	mini-prepped RecA1 Ecor1 Pst1 mutegensis cultures A and B &lt;/br&gt;
•	sent the test to sequencing. Terminator came out but the driver didn’t show up. Remu did not e &lt;/br&gt;
•	going to  assemble test again using B0015 mini-prep from 7/14 going to PCR driver from registry &lt;/br&gt;
•	also going to try to assemble with Terminator as the insert and the driver as the vector. Terminator insert digest with E&amp;S enzymes and Vector digest with E&amp;X &lt;/br&gt;
 &lt;/br&gt; &lt;/br&gt;
7/19 &lt;/br&gt;
•	test using mini-prep from 7/14 of terminator and registry assembled with terminator as vector and driver as insert &lt;/br&gt;
•	PCR driver  &lt;/br&gt;
•	Digest &lt;/br&gt;
•	Ligate &lt;/br&gt;
•	Transformed  &lt;/br&gt;
o	Time constant 2.0 &lt;/br&gt;
•	Plated &lt;/br&gt;
•	Also assembled mini-prep terminator and mini-prep driver 7/14 but with terminator as insert and driver as insert &lt;/br&gt;
•	PCR terminator &lt;/br&gt;
•	Digest using buffer 4 &lt;/br&gt; 
•	Ligate &lt;/br&gt;
•	Transformed &lt;/br&gt;
o	T.C. 2.0 &lt;/br&gt;
•	Plated A&amp;B with assemble of terminator as insert and driver as vector &lt;/br&gt;
•	Plated C&amp;D with assemble of terminator as driver and driver as vector &lt;/br&gt;
RecA1 Mutagenesis &lt;/br&gt;
•	Multisite mutagenesis was conducted for EcoR1 and Pst1 &lt;/br&gt;
•	Protocol followed was the desired mutagenesis from open wetware &lt;/br&gt; 
•	Also ran mutagenesis adjusting the original multisite muteagenesis protocol &lt;/br&gt;
o	MgSO4: 0, 0.5, 1 ul &lt;/br&gt;
o	DMSO: 0, 0.5, 1  &lt;/br&gt;
o	Without DMSO with primer &lt;/br&gt;
•	Let PCR run overnight &lt;/br&gt;
Mutagenesis—multisite protocol varying MgSO4 and addition of Reverse Primer: &lt;/br&gt;
•	With reverse primer &lt;/br&gt;
o	1: 0ul MgSO4  &lt;/br&gt;
o	2: 0.5ul MgSO4  &lt;/br&gt;
o	3: 1ul MgSO4 &lt;/br&gt;
•	Without reverse Primer  &lt;/br&gt;
o	4: 0ul MgSO4  &lt;/br&gt;
o	5: 0.5ul MgSO4  &lt;/br&gt;
o	6: 1ul MgSO4 &lt;/br&gt;
•	Due to supplies was only able to do one mutagenesis, choose to do 4 &lt;/br&gt;
 &lt;/br&gt; &lt;/br&gt;
7/20 &lt;/br&gt;
•	PCR colony tests &lt;/br&gt;
•	Cultured test &lt;/br&gt;
RecA &lt;/br&gt;
•	Incubated 4 hours &lt;/br&gt;
•	PCR purified RecA1 mutagenesis  &lt;/br&gt;
•	Transformed  &lt;/br&gt;
o	T.C. 4.4 &lt;/br&gt;
•	Plated &lt;/br&gt;
•	Reviewed sequencing results from 7/18, came back unsuccessful &lt;/br&gt;
 &lt;/br&gt; &lt;/br&gt;
7/21 &lt;/br&gt;
•	Mini-prep RecA1 send to sequencings  &lt;/br&gt;
•	Culture RecA1 mute1  &lt;/br&gt;
•	Mini-prep terminator and driver and RecA1 III &lt;/br&gt;
•	Also made freezer stock &lt;/br&gt;
•	PCR insert of terminator with extension time 45 seconds  &lt;/br&gt;
•	Plated two colonies (I, II) from transformation on 7/19 (PCR without reverse primer) &lt;/br&gt;
•	Made culture &lt;/br&gt;
 &lt;/br&gt; &lt;/br&gt;
7/22 &lt;/br&gt;
•	No centrifuge tubes so unable to send to sequencing &lt;/br&gt;
•	PCR purified terminator &lt;/br&gt;
•	Digested terminator as insert and driver as vector &lt;/br&gt;
•	Ligated &lt;/br&gt;
•	Transformed &lt;/br&gt;
o	T.C. 1.8 &lt;/br&gt;
•	Mini-prepped culture (I and II) from 7/21 and sent to sequencing &lt;/br&gt;
•	Began other mutagenesis’s by beginning of PCR &lt;/br&gt;
o	1, 2, 3, 5, and 6 &lt;/br&gt;
 &lt;/br&gt; &lt;/br&gt;
7/23 &lt;/br&gt;
•	Incubated Mutagenesis’s for 4 hours &lt;/br&gt;
•	PCR purified &lt;/br&gt;
•	Transformed &lt;/br&gt;
o	T.C. &lt;/br&gt;
	1: 4.4 &lt;/br&gt;
	2: 4.8 &lt;/br&gt;
	3: 4.8 &lt;/br&gt;
	5: 4.8 &lt;/br&gt;
	6: 4.0 &lt;/br&gt;
 &lt;/br&gt; &lt;/br&gt;
7/24 &lt;/br&gt;
•	Colonies were not very big waiting until Monday  &lt;/br&gt;
•	Began another RecA mutagenesis starting with PCR &lt;/br&gt;
o	Primers Pst, Arg243, Lys 286 &lt;/br&gt;
•	Minipreped mutagenesis 4 and sent to sequencing &lt;/br&gt;
 &lt;/br&gt; &lt;/br&gt;
7/25 &lt;/br&gt;
•	Cultured colonies from test 1A, 1B, 2A, 2B, 3A, 3B, 5A, 5B, 6A, ^B and test &lt;/br&gt;
•	Reviewed sequencing: unsuccessful  &lt;/br&gt;
•	Incubated second RecA mutagenesis &lt;/br&gt;
•	PCR purified &lt;/br&gt;
•	Transformed &lt;/br&gt;
 &lt;/br&gt; &lt;/br&gt;
7/26 &lt;/br&gt;
•	Minipreped colonies however was late to sequencing  &lt;/br&gt;
•	Began a third mutageneis with different amounts of DMSO and PCR annealing temperature &lt;/br&gt;
 &lt;/br&gt; &lt;/br&gt;
7/27 &lt;/br&gt;
•	Cultured test again &lt;/br&gt;
•	Sequencing results had a lot of Ns &lt;/br&gt;
•	Started new test assembly &lt;/br&gt;
o	1: Vector = Driver Enzymes E&amp;X, Insert=Terminator, Enzymes E&amp;S &lt;/br&gt;
o	PCRed, ligated, transformed (T.C. 2.4) palted &lt;/br&gt;
•	Incubated RecA &lt;/br&gt;
•	PCR purfified &lt;/br&gt;
•	Transformed (T.C: 5.2) &lt;/br&gt;
•	plated &lt;/br&gt;
 &lt;/br&gt; &lt;/br&gt;
7/28 &lt;/br&gt;
•	Pst primer and EcoR1 primers were re-ordered because discovered they were incorrect &lt;/br&gt;
•	No colonies grew on test plate &lt;/br&gt;
•	Grew terminator and driver from freezer stock &lt;/br&gt;
 &lt;/br&gt; &lt;/br&gt;
7/29  &lt;/br&gt;
•	Cultured RecA from plates &lt;/br&gt;
•	Mini-prepred terminator and driver and continued assembly (T.C. 3.2) &lt;/br&gt;
•	Mini-prep RecA mutagenesis and sent to sequencing  &lt;/br&gt;
 &lt;/br&gt; &lt;/br&gt;
7/30  &lt;/br&gt;
•	cultured test assembly &lt;/br&gt;
•	reviewed RecA results: unsuccessful &lt;/br&gt;
 &lt;/br&gt; &lt;/br&gt;
7/31 &lt;/br&gt;
•	started new RecA mutagenesis &lt;/br&gt;
o	varying concentrations and PCR temperatures &lt;/br&gt;
•	mini-prep test and sent to sequencing &lt;/br&gt;
 &lt;/br&gt; &lt;/br&gt;
8/1 &lt;/br&gt;
•	continued RecA mutagenesis &lt;/br&gt;
•	reviewed test sequencing  &lt;/br&gt;
 &lt;/br&gt; &lt;/br&gt;
8/2 &lt;/br&gt;
•	reviewed test and RecA mini-preps using a nanodrop because suspicion of bad sequencing read &lt;/br&gt;
•	several did not turn out well so re-mini-prepped and sent to sequencing &lt;/br&gt;
 &lt;/br&gt; &lt;/br&gt;
8/3 &lt;/br&gt;
•	checked sequencing still unsuccessful &lt;/br&gt;
 &lt;/br&gt; &lt;/br&gt;
8/4 &lt;/br&gt;
•	discovered mini-prep test kit was contaminated &lt;/br&gt;
•	retrieved new kit and began mutagenesis  &lt;/br&gt;
 &lt;/br&gt; &lt;/br&gt;
8/8 &lt;/br&gt;
•	Began test assembly &lt;/br&gt;
•	Using terminator and driver from 7/18 (had be verified through sequencing that it was terminator and driver)  &lt;/br&gt;
•	Digest &lt;/br&gt;
o	Driver as vector enzymes E&amp;X &lt;/br&gt;
o	Terminator as insert E&amp;S  &lt;/br&gt;
•	Ligated &lt;/br&gt;
•	Transformed ligation  &lt;/br&gt;
o	T.C.: 2.2 &lt;/br&gt;
•	Plated &lt;/br&gt;
 &lt;/br&gt; &lt;/br&gt;
8/9 &lt;/br&gt;
•	Cultured parts for new assembly &lt;/br&gt;
•	Colony PCR product from 8/8 &lt;/br&gt;
 &lt;/br&gt; &lt;/br&gt;
8/12  &lt;/br&gt;
•	Cultured test, terminator and driver &lt;/br&gt;
 &lt;/br&gt; &lt;/br&gt;
8/13 &lt;/br&gt;
•	Miniprepped from 8/12 &lt;/br&gt;
 &lt;/br&gt; &lt;/br&gt;

9/8&lt;/br&gt;
-	Using CBAR for mutagenesis &lt;/br&gt;
-	Designed oligos  -&gt; ordered&lt;/br&gt;
-	PCR’d boh B0015 and I763007&lt;/br&gt;
&lt;/br&gt;&lt;/br&gt;
9/9&lt;/br&gt;
-	Oligo arrived -&gt; ran PCR&lt;/br&gt;
-	Rec A Lys Mutation&lt;/br&gt;
o	Pst Forward, EcoR1 Reverse&lt;/br&gt;
o	Pst R, EcoR1 F&lt;/br&gt;
-	RecA Pst Mutagenesis&lt;/br&gt;
o	Arg F, Lys R&lt;/br&gt;
o	Arg R, Lys F&lt;/br&gt;
-	PCR Purified – B0015 and I763007&lt;/br&gt;
-	Digested&lt;/br&gt;
o	Term&lt;/br&gt;
	Vector = S, P&lt;/br&gt;
	Insert = E, S&lt;/br&gt;
o	Driver&lt;/br&gt;
	Vector = E, X&lt;/br&gt;
	Insert = X, P&lt;/br&gt;
-	Digest Purified Vectors&lt;/br&gt;
-	Ligated&lt;/br&gt;
o	Test 1 = Term (V) + Driver (I)&lt;/br&gt;
o	Test 2 = Term (I) + Driver (V)&lt;/br&gt;
-	Transformed&lt;/br&gt;
o	Test 1 = 3.0&lt;/br&gt;
o	Test 2 = 3.0&lt;/br&gt;
-	Plated&lt;/br&gt;
o	A plates&lt;/br&gt;
&lt;/br&gt;&lt;/br&gt;
9/12&lt;/br&gt;
-	PCR Purified&lt;/br&gt;
-	Nanodropped&lt;/br&gt;
o	14.6 ng/µL&lt;/br&gt;
o	19.8&lt;/br&gt;
o	41.5&lt;/br&gt;
o	9.7&lt;/br&gt;
-	Fentamole Calculations were done&lt;/br&gt;
-	Waited to verify calculation with supervisor&lt;/br&gt;
-	Diluted PCRs to correct concentration &lt;/br&gt;
-	Combined the following together w/ CBAR mixture:&lt;/br&gt;
o	Pst F + EcoR1 R with Pst R + EcoR1 F&lt;/br&gt;
o	Arg F + Lys R with Arg R + Lys F&lt;/br&gt;
-	Incubated at 50 degrees C for 1 hour.&lt;/br&gt;
-	PCR purified&lt;/br&gt;
-	Transformed .5 µL on K resistance&lt;/br&gt;
-	Plated&lt;/br&gt;
-	PCR Colony – Test 1 and Test 2&lt;/br&gt;
-	Ran on gel&lt;/br&gt;
o	Looked good&lt;/br&gt;
-	Cultured&lt;/br&gt;
-	Transformed 2nd half of circuit&lt;/br&gt;
o	T.C. =5.2&lt;/br&gt;
-	Cultured part&lt;/br&gt;
&lt;/br&gt;&lt;/br&gt;
9/13&lt;/br&gt;
-	Miniprepped test 1, 2, and the second half of the circuit (B)&lt;/br&gt;
-	Sent to sequencing&lt;/br&gt;
&lt;/br&gt;&lt;/br&gt;
9/14&lt;/br&gt;
-	Colony stab and cultured&lt;/br&gt;
-	Read sequencing results – Test 1, 2, B&lt;/br&gt;
o	Test 1 = not good&lt;/br&gt;
o	Test 2 = GOOD! :D&lt;/br&gt;
o	Test B = GOOD! :D&lt;/br&gt;
o	PCR’d all parts that worked&lt;/br&gt;
&lt;/br&gt;&lt;/br&gt;
9/15&lt;/br&gt;
-	CBAR Epic fail&lt;/br&gt;
-	Ran PCR product on gel&lt;/br&gt;
o	Looked good&lt;/br&gt;
-	PCR Purified&lt;/br&gt;
-	Digested&lt;/br&gt;
o	Part 1 (Test 2)&lt;/br&gt;
	Vector = S,D&lt;/br&gt;
	Insert = E, S&lt;/br&gt;
o	Part 2 (Test B)&lt;/br&gt;
	Vector = E, X&lt;/br&gt;
	Insert = X, P&lt;/br&gt;
-	Ligated&lt;/br&gt;
o	Test 1 = Part 1 (V) + Part 2 (I)&lt;/br&gt;
o	Test 2 = Part 2 (V) + Part 1 (I)&lt;/br&gt;
-	Transformed&lt;/br&gt;
o	Test 1 = 2.8&lt;/br&gt;
o	Test 2 = 2.6&lt;/br&gt;
-	Plated&lt;/br&gt;
o	A Resistence&lt;/br&gt;
&lt;/br&gt;&lt;/br&gt;
9/16&lt;/br&gt;
-	Test 1 nor Test 2 grew – started over&lt;/br&gt;
9/19&lt;/br&gt;
-	Mutagenesis&lt;/br&gt;
o	PE + Arg -&gt; fail&lt;/br&gt;
o	PE + Lys -&gt; worked&lt;/br&gt;
o	LE + Arg -&gt; fail&lt;/br&gt;
o	LE + Pst1 -&gt; fail&lt;/br&gt;
&lt;/br&gt;&lt;/br&gt;


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==Monday, May 30==
===RecAI Extraction, Day 1===
The RecA group extracted the RecAI gene from Escherichia coli using the genomic DNA extraction kit produced by Omega Bio-Tek. The RecA group created a culture of the RecAI gene and left this culture to grow overnight.
[[File:RecA designs.jpg|right|thumb|400px|Figure 1: The overview for RecAI mutagenesis.]]
==Tuesday, May 31==
===RecAI Extraction, Day 2===
The RecA group performed miniprep on the RecAI culture from 5/30/11. The RecA group placed the RecAI gene into the C3 vector through amplified insert assembly in order to perform mutagenesis on the RecAI gene. This means restriction digest, followed by ligation, transformation into E. coli cells and plating to grow colonies over night.
===RecA Mutagenesis Test, Day 1===
The RecAI team also developed a plan for RecA mutagenesis. Figure 1 shows the overview of this plan. The following table describes each part in more detail.
{|border=&quot;1&quot;
!Part
!Registry Name
!Resistance
!Position on Registry Plate
!Length (bp)
!Digested as (insert/vector)
|-
|1: Terminator
|B0015
|A/K
|Plate 1&lt;br /&gt;Well 23L
|129
|Vector
|-
|2: Reporter (cI/RFP)
|I763007
|A/A
|Plate 1&lt;br /&gt;Well 15J
|918
|Insert
|-
|3: Constitutive&lt;br /&gt;Promoter
|J23118
|A/A
|Plate 1&lt;br /&gt;Well 22A
|35
|Vector
|-
|4: cI Repressor
|K081007
|A/A
|Plate 2&lt;br /&gt;Well 20J
|796
|Insert
|}

The RecA group plans to insert RecA mutants downstream of the cI Repressor so that the constitutive promoter will continuously transcribe the cI Repressor and the RecA mutant. If the mutant has recombinant activity then the reporter will be deleted because of the homologous sequences of the Terminator (Part 1) and the terminator of the Reporter due to homologous sequences of the Terminator (Part 1) and the terminator of the Reporter, part 2 will be deleted.

==Wednesday, June 1==
===RecAI Extraction, Day 3===
The RecA group ran colony PCR for the RecAI+C3 constructs created 5/31/11. The agarose gel electrophoresis test showed that the assembly worked.
===RecAI Mutagenesis Test, Day 2===
The RecA group received their ordered parts from the registry and performed amplified insert assembly to add B0015 and I763007 together, as well as J23118 and K081007 together. This ended with allowing colonies to grow overnight on plates of the correct resistance.

==Thursday, June 2==
===RecAI Extraction Take 2, Day 1===
The RecA group realized that the RecAI+C3 colony dried up, so the group needed to make another one, starting with culturing RecAI from genomic DNA.
===RecAI Mutagenesis Test, Day 3===
The RecA group made cultures of the B0015+I763007 construct and the J23118+K081007 construct.

==Friday, June 3==
===RecAI Extraction Take 2, Day 2===
The RecA group miniprepped RecAI and assembled RecAI and C3 through amplified insert assembly.
===RecAI Mutagenesis Test, Day 3===
The RecA group prepared their assembly for sequencing by performing the miniprep procedure on the B0015+I763007 and the J23118+K081007 constructs. The RecA group then performed amplified insert assembly to combine the previous two constructs into one plasmid. The J23118+K081007 served as the insert and the B0015+I763007 served as the vector.

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