Team:Harvard/allergy/notebook

(Difference between revisions)

notebook calendar

Week 1	06-14-2010	06-15-2010	06-16-2010	06-17-2010	06-18-2010
Week 2	06-21-2010	06-22-2010	06-23-2010	06-24-2010	06-25-2010
Week 3	06-28-2010	06-29-2010	06-30-2010	07-01-2010	07-02-2010
Week 4	07-05-2010	07-06-2010	07-07-2010	07-08-2010	07-09-2010
Week 5	07-12-2010	07-13-2010	07-14-2010	07-15-2010	07-16-2010
Week 6	07-19-2010	07-20-2010	07-21-2010	07-22-2010	07-23-2010
Week 7	07-26-2010	07-27-2010	07-28-2010	07-29-2010	07-30-2010
Week 8	08-02-2010	08-03-2010	08-04-2010	08-05-2010	08-06-2010
Week 9	08-09-2010	08-10-2010	08-11-2010	08-12-2010	08-13-2010

06-14-2010 [ top ]

06-15-2010 [ top ]

06-16-2010 [ top ]

06-17-2010 [ top ]

06-18-2010 [ top ]

06-21-2010 [ top ]

• PCR amplification of gDNA from Arabadopsis thaliana for sense and antisense parts of LTP, Bet v 1, and Ger3.
• Diagnostic Digest of PCR products

Results

Lane 2 is LTP sense, lanes 11 and 12 are Ger 3 sense and antisense.

06-22-2010 [ top ]

• Gel electrophoresis of PCR products
• Gel digest and purification of LTP sense, Ger sense and Ger antisense
• Digest with Xba and Pst
• Purification of parts

Results
Gel purification yielded 2.6, 7.8, and 7.9 ng/μL of LTP sense, Ger sense, and Ger antisense parts. We were able to obtain 1.5, 4.3 and 0.5 ng/μL of digested LTP sense, Ger 3 sense, and Ger 3 antisense, respectively, from the digest purification.

06-23-2010 [ top ]

• Digest and purification of vector V0120
• Ligation of LTP sense, Ger sense, and Ger antisense parts into V0120

Results
Ligations were plated.

06-24-2010 [ top ]

06-25-2010 [ top ]

06-28-2010 [ top ]

06-29-2010 [ top ]

06-30-2010 [ top ]

07-01-2010 [ top ]

• Ligations for full amiRNA and V0120 (w/o death gene)

note: .5 denotes multistep pcr reaction from yesterday in which .5 uL of part 3 of gfp/ltp were use in one of the giant assemblies of parts 1,2,3 w/ primers A&B

Ligation Reactions

	Bet	GFP	LTP	LTP(.5)	GFP(.5)
DNA Insert (after 1:10 dilutions)	3	4.5	3	4.71	4.1
T4 DNA ligase buffer (10x)	2	2	2	2	2
diH2O	11.5	10	11.5	9.79	10.4
T4 DNA ligase	1	1	1	1	1
DNA Backbone	2.5	2.5	2.5	2.5	2.5

• • Used 50 ng of backbone and 150 ng of insert (ratio of insert to vector ~.14
  • Did 1:10 dilutions for the digested inserts

• PCR of PME3 and PAL2
  • saw no bands from last night's pcr so we re ran it

Annealing Temp: 69C Extension Time: 30 sec

2 reactions (PME & PAL)

Name	Amount (uL)
10mM dNTPs	1
5x Phusion Buffer	10
Phusion Polymerase	.5
Arabadopsis Genomic DNA	1
Fwd/Rev Primer	.5 uM each (1uL 50x)
Water	35.5

Concentrations: PME: 250 ng/uL PAL: 134 ng/uL

• gel: lane 1:ladder; lane 2: Pal3; lane 3: Pme 2; lane 4: ladder

• Sent amiRNA Bet for sequencing

• Ligation of Antisense/sense allergens into V0120 (lacking death gene)

Ligation Reactions

	BetS	BetA	LTPS	LTPA	GerS	GerA	Bet 1.2S	Bet 1.2 A	Negative Control
DNA Insert (after 1:10 dilutions) ~ 3 times excess	3	3	3	3	3	3	3	3	0
T4 DNA ligase buffer (10x)	2	2	2	2	2	2	2	2	2
diH2O	12	12	12	12	12	12	12	12	15
T4 DNA ligase	1	1	1	1	1	1	1	1	1
DNA Backbone	2	2	2	2	2	2	2	2	2

• • Used 34 ng of backbone and 10 ng of insert (ratio of insert to vector ~.09375

07-02-2010 [ top ]

• Check on sequences for Bet
• Digest Pme3 and Pal 2 introns/Ligate w/ V0120/Transform
  • Digestions

Digestion Reactions

	PME 3 (4 digestions)	Pal 2 (4 digestions)	Backbone (B21 w/ YFP)
DNA	4 (~1000ng)	7 (~1000ng)	3 (~900 ng)
FD Buffer (10x)	2	2	2
diH2O	12	9	13
EcoRI	1	1	1
SpeI	1	1	1

• • PCR purified digests
    • Concentrations: Pme (ranges from 20-36 ng/uL); Pal (ranges from 20 to 28 ng/uL)
  • Ran backbone on gel and gel purified (Concentration: 3.1 ng/uL)

• Ligations of Pme/Pal w/ V0120

Ligation Reactions

	Pme4	Pal3	Pme5	Pal1
DNA Insert	1.85	1	1	4.86 after 1 in 10 dilution
T4 DNA ligase buffer (10x)	2	2	2	2
diH2O	14.15	15	6.3	--
T4 DNA ligase	1	1	1	1
DNA Backbone	1 (bbone 2)	1 (bbone 2)	14	9.64

• • bbone 2 was potentially contaminated at a higher concentration 96.1 ng/uL

• Check on transformations of amiRNA (5 transformations) and antisense/sense allergens (8 transformations)
  • There were colonies from the amiRNA transformations
    • We grew up 24 colonies (3 for each GFP, GFP .5, LTP, LTP .5, BET) ~ 10 am to be ready ~3 pm for minipreps)
    • The other 8 transformations did not show any colonies--we put it back in to see if we would get colonies (only 100 uL instead of 200 uL was plated)
    • To do: Grow up colonies to miniprep, run on gel, sequence

• pHannibal and pKannibal arrived today (hannibal is amp resistant and kannibal is kan resistant)
  • We transformed these plasmids

07-05-2010 [ top ]

07-06-2010 [ top ]

Team Allergy

Today, we isolated plasmid that contained antisense and sense sequences of the allergen panel and introns PME and PAL that we grew last week.

Procedures

For amiRNA

1. Nanodrop of miniprepped amiRNA plasmids
2. Diagnostic digest of amiRNA plasmids
3. E Gel of digested amiRNA
4. Send plasmids to Genewiz for sequencing

For ihpRNA

1. Miniprep of plasmids containing introns for hpRNA
2. Nanodrop of miniprepped intron plasmids
3. Miniprep of plasmids containing Allergen Panel sense and antisense
4. Nanodrop of minipreped allergen plasmids
5. Diagnostic Digest of introns and allergens
6. E Gel of digested parts
7. Extract pKannibal and pHannibal vectors and transform into E. coli
8. PCR of the PDK intron in pKannibal and pHannibal

Results

For amiRNA

Miniprep of turbo bacteria for plasmids containing sequences for amiRNA

Should obtain plasmids containing the complete sequence for amiRNA. The resulting concentration should be relatively high (<100ng/μL) because all growing bacteria would have the plasmids inside.

Nanodrop of miniprepped amiRNA plasmids

We used 2μL of DNA per nanodrop.

Concentrations in ng/μL

Sample # -->	One	Two	Three
GFP	134.4	167.6	207.2
GFP 0.5	163.3	106.9	230.9
LTP	134.0	127.8	165.5
LTP 0.5	158.5	137.2	113.5
Bet	129.4	78.9	124.4

Diagnostic digest of amiRNA plasmids Digested 200~500ng of plasmids with EcoR1 and Spe1. We will then run the digested vectors on an E Gel and look for DNA that is the length of the insert. If such DNA appears on the gel, then we will send it off to be sequenced.

E Gel of digested amiRNA

We had to run 21 samples, so we used two gels. The orders were:

Gel #1

1. 1kb Ladder
2. GFP 1
3. GFP 2
4. GFP3
5. GFP 0.5 1
6. GFP 0.5 2
7. GFP 0.5 3
8. LTP 1
9. LTP 2
10. LTP 3
11. LTP 0.5 1 no visible band for neither plasmid nor insert
12. LTP 0.5 2

Gel #2

1. 1kb Ladder
2. LTP 0.5 3
3. Bet 1
4. Bet 2
5. Bet 3
6. Neg Control for hpRNA (digested with Xba and Pst, and ligated without new inserts) no visible insert band
7. Neg Control for intron (digested with Eco and Spe, and ligated without new inserts)
8. PME Good no visible insert band
9. PME Dubious no visible insert band
10. PAL Good colony 1 no visible insert band
11. PAL Good colony 2
12. 1kb Ladder

For ihpRNA

Miniprep of plasmids containing introns for hpRNA Nanodrop of miniprepped intron plasmids

We used 2μL of DNA per nanodrop.

Concentrations in ng/μL, cells are filled when applicable

Insert Name	Good	Dubious	Colony 1	Colony 2	Colony 3
PME	85.6	66.8
PAL			67.7	108.8
Neg Control, Intron	74.0
Neg Contron, hpRNA			63.5	30.1	37.3

1. Miniprep of plasmids containing Allergen Panel sense and antisense (from overnight cultures)--still need to do preps of cultures grown today
2. Nanodrop of minipreped allergen plasmids
3. Diagnostic Digest of introns and allergens
4. E Gel

Gel #1

Lane 1: 1kb Ladder

Lanes 2-4: Ger Sense

Lanes 5-7: Ger Antisense

Lanes 8-10: LTP Sense

Lanes 11-13: LTP Antisense

Lanes 14-16: Bet 2 Sense

Lanes 17-19: Bet 2 Antisense

Lanes 20-22: Bet 1 Sense

Lanes 23-25: Bet 1 Antisense

phannibal and pkannibal Extracted vectors from a piece of paper per instructions. Transformed vectors into E. coli. Miniprep and PCR to extract PDK intron.

• pcr to get out pdk intron

07-07-2010 [ top ]

07-08-2010 [ top ]

07-09-2010 [ top ]

07-12-2010 [ top ]

07-13-2010 [ top ]

07-14-2010 [ top ]

07-15-2010 [ top ]

07-16-2010 [ top ]

07-19-2010 [ top ]

07-20-2010 [ top ]

07-21-2010 [ top ]

07-22-2010 [ top ]

07-23-2010 [ top ]

07-26-2010 [ top ]

07-27-2010 [ top ]

07-28-2010 [ top ]

07-29-2010 [ top ]

07-30-2010 [ top ]

08-02-2010 [ top ]

08-03-2010 [ top ]

• Grew up cultures of completed ihpRNA constructs (Bet, LTP, Ger) in pORE expression vector
• amiRNA PCR
  • Will look at results of PCR tommorrow

08-04-2010 [ top ]

Tasks

• amiRNA PCR appears to have worked at every Tm we tried:

• Digested V9/V10 to insert our constructs into
  • Realized that we hadn't gel purified our ihpRNA inserts
  • Gel purification of inserts (entire ihpRNA parts)

Ladder, 9, 11c1, 11c2, ladder, 25c1, 28c1, 28c2, 36c1, 36c2, ladder

Results

• Successfully gel purified our inserts and digested backbones that we will ligate into

08-05-2010 [ top ]

• Gel extracted V9/V10 backbone

Lanes: Ladder, V9, Ladder, V10

Concentrations: V9 (9.4 ng/uL; V10 (16.4 ng/uL)

• Ligated ihpRNA inserts into V9/V10 and transformed
  • For our ligations we only used ~ 2uL of backbone (around 18 and 32 ng of backbone)and used a 3x excess of insert
• Verified that amiRNA stitching of Bet, LTP yielded the proper insert with a low level of background through PCR:

Lanes: 1-7: Bet, corresponding to 65.55 degrees C for stitching Tm (even spacing)

Lanes: 8-10: LTP, corresponding to 65.55 degrees C during stitching annealing, even spacing

• Digested
  • Bet,LTP inserts with X+P; B21 with X+P+phosphatase
• Ligated, Transformed

08-06-2010 [ top ]

amiRNA

• amiRNA-transformed turbo cells didn't grow in YEB medium. There should be more LB+Amp available by the end of the day. In the meantime, we're digesting some more backbone (8x20ul X+P+B21)
  • ligations of Bet/LTP into V0120

ihpRNA

• no colonies on plates from transformations done yesterday after 16 hours (we let these plates grow for longer)
• redoing ligations of ihpRNA inserts in V9/V10
  • Redigested and gel purified some backbone

• Concentrations: V9 (5 ng/uL); V10 (6 ng/uL)
  • 12 ligations each for V9 and V10

08-09-2010 [ top ]

• DTLs of amiRNA insert were unsuccessful. Yesterday we concluded this was because turbo cells + amp resistance don't grow on YEB media (we were out of LB+amp media). Today we must adopt a new hypothesis as 0/16 colonies grew on LB+amp media. Working theory is that the backbone digestions pictured below have the digested backbone and inserts as the two bands (top to bottom) instead of the undigested & digested backbone (in hindsight, I was trying to ligate into YFP*2, the original insert of B21). The confusion arose from the 1kb ladder, which at first appeared to match another ladder far better than 1kb (see Thursday's entry or thereabouts). This, combined with the fact that the 1kb ladder was hand-labeled, led me to suspect that it was in fact the other ladder (pR322 as pictured [http://openwetware.org/wiki/Image:Ladders.jpg here]). This, in turn, supported the (theorized incorrect) conclusion that the top & bottom bands [http://openwetware.org/wiki/Image:Bbcut1.jpg here] and [http://openwetware.org/wiki/Image:Bbcut2.jpg here] were undigested & digested backbone, respectively, and that the insert had diffused. I've DTL'd again, this time using the upper band (which didn't separate into digested & undigested bands after running for 30m on a .7% agarose gel) for ligation. We'll see on Monday how it went.

• New Plan of action:

1. Redigest V9/V10 (three digests of each backbone to use more DNA in ligations)
2. Grew up V9/V10+ ihpRNA colonies
3. Re pcrd amiRNA and digested to ligate into V9/V10
4. Catalog parts to be ready for the registry

08-10-2010 [ top ]

ihpRNA

• Only 2 colonies grew up (V10 9 (37c1) and V10 12 B(38 c2)) ger ihprna constructs
  • growing up colonies again in 5 mL cultures w/less antibiotic (50 ng/uL of kan)
  • concentrations: V10_9: 21.4 ng/uL V10_12B: 16.9 ng/uL
• Diagnostic Digest (Should see ~ 7 kb band and 840 band)

• Did not see exepcted bands (waiting on cultures grown up today to miniprep and hopefully transform into agrobacterium tommorow)

amiRNA

• Ligated into V9/V10 and transformed

08-11-2010 [ top ]

• Minipreps of (V95, V91, V99, V99b, V1011, V107B, V1012, V109b)
  • Concentrations (20-60 ng/uL range)
• Diagnostic Digest (should see ~ 7 kb and 840 bp)

Ladder, V9 5 (Bet2), V91(LTP), V99 (Ger), V99B (Ger), V1011 (Ger), V107b(Ger), V1012 (Ger), V109B (Ger)

V10 7B (Ger worked)

08-12-2010 [ top ]

ihpRNA

• Minipreps of more V9/V10 colonies
• Diagnostic Digest of minipreps w/ Eco and Pst (Should see ~840 and 7000 bp)

ladder, V92, V92b, V94, V94b, V95, V96b, V97, V97b, V98, V98b

Lanes 2&3: LTP (11c1); Lanes 4&5: Bet2 (25c1); Lane 6: Bet2 (25c2); Lane 7: Bet2 (28c1); Lanes 8&9: Ger (36c1); Lanes 10&11 Ger (36c2)

• Digested V9/V10 backbone for ligations w/ Eco & Pst (redoing ihpRNA ligations into V9/V10)

amiRNA

• Ligated into V9/V10 and transformed (mutagenesis sites confirmed through pcr using 20 bp sepecific primers)
  • still need to be sequenced

08-13-2010 [ top ]

• Organized parts so we could find them when we came back

<Note: remember to add more pics/less text>