Identification of Plasmid Code: 4118B25 Kayson Lambert

A Plasmid is a circular piece of a certain be cut by restriction en"ymes ,g *, P!u*, and then ran a single digest using P!u*, and a gene of DNA, which can be inserted into a cell to Pst*. make that cell exhibit the trait that the plasmid codes for. n order to determine what plasmid was gi!en, first had to disco!er the si"es of all

double digest with ,g * and Pst*. %he gel used was a .+1 gel, meaning that in a .2 ml gel, used . ) grams of agarose solution, which is optimal for

possible options that were gi!en to me, then run a DNA fragments between +22 and *-222 base pairs. digest. After running my &el 'lectrophoresis, then #hen you digest DNA, you use $estriction obser!ed the results, recording distances tra!eled, en"ymes to cut it into certain si"es of DNA and using the ladder determined base pair si"es,

fragments. %hen you can run a &el 'lectrophoresis and ha!e determined my Plasmid code3 to determine the si"es of these fragments. (rom )**+,,-. is p,L0.

there you can compare the si"es you were supposed Methods to get from the digest to the actual numbers you recei!ed from the gel. %o start my experiment, first used New 'ngland ,iolab4s online !irtual digest, N',5utter,

%his method is !ery useful if you know you to determine the !alues that should see from a only ha!e a few options that your unknown could single digest3 be, but it is !ery effecti!e at ruling out options, and pAMP narrowing in on the plasmid you actually ha!e. Abstract was told that my plasmid code )**+,-. would be either pA/P, pKAN, or p,L0. ran a
pKA pB$% fragment sizes (bp) with BgI1 158, 1118, 3263 26!, "#!, 313# 15"6, 1"!&, 2121

pAMP pKA pB$%

fragment sizes (bp) with P'(1 8#6, 36!3 5"2, 3622 !53, !8&, "26, 1"#8, 1#8&
fragment sizes (bp) with Pst1 !53# #23, 32"1 1#", 1316, 3#2!

!irtual digest to obser!e how each of these would

Identification of Plasmid Code: 4118B25 Kayson Lambert

placed my DNA fragments approximately * inch
pAMP pKA pB$%

from the end of the gel, at farthest. Afterwords used an 0ltra:iolet light to !iew where my DNA fragments were, and using a

6r a double digest3
pAMP pKA pB$% fragment sizes (bp) with BgI1 ) Pst1 158, 1118, 1185, 2&"8 261, 313, "#!, #23, 1#&3 125, 188, 1#2, 11#1, 1615, 2121

printer, printed - copies of the image Results #hen !iewing the distances, and subse7uently DNA fragment si"es that my plasmid

After determining what my results should be,

broke up into, ha!e concluded that ha!e p,L0.

calculated the amount of agarose re7uired to make ;(ig *<, as that was the only plasmid which had the correct 1 of a .2 ml gel. determine my digest recipe3
*(be 1/ntr/+ P'(1 BgI1)Pst1 p+asmi, (-+) 1&. b(ffer 3 (-+) ,223 (-+) 2 2 2 2 2 2 P'(1 (-+) 16 15 1! & 1 & BgI1 (-+) & & 1 Pst1 (-+) & & 1 */ta+ 0/+(me (-+) 2& 2& 2&

then proceeded to

predicted si"es similar to my results.

Conclusion prepared each sample in a *.. mL micro8 centrifuge tube, and then put them on ice for the next week. %his Lab was designed to test my ability to determine what an unknown plasmid was, and so

then made my gel, and adding ladder did. #hile my numbers were not as exact as

on either end of the gel, loaded my samples in the would like them to be, causing a little uncertainty following order3 5ontrol P!u* ,g *9Pst* in my results, ha!e pro!en my ability to do so. belie!e that a ma=or source of error could ha!e been contamination before ac7uired the sample,

ran my gel at *)2 !olts for )2 minutes, which

Identification of Plasmid Code: 4118B25 Kayson Lambert

as all of the optional plasmids had base pairs smaller that about .222, and yet was recei!ing some numbers abo!e *2222, which has led me to conclude it must ha!e been contaminated at some point before recei!ed it.

+a,,er ban, 1 +a,,er ban, 2 +a,,er ban, 3 +a,,er ban, ! +a,,er ban, 5 +a,,er ban, 6 +a,,er ban, " +a,,er ban, 8 +a,,er ban, # 6/ntr/+ ban, 1 6/ntr/+ ban, 2 6/ntr/+ ban, 3 6/ntr/+ ban, ! P'(1 ban, 1 P'(1 ban, 2 P'(1 ban, 3 P'(1 ban, ! P'(1 ban, 5 BgI1 )Pst1 ban, 1 BgI1 )Pst1 ban, 2 BgI1 )Pst1 ban, 3 BgI1 )Pst1 ban, ! BgI1 )Pst1 ban, 5 BgI1 )Pst1 ban, 6

Ban, Migrati/n (mm) 4ragment 5ize (bp) !1 1&&&& !5 8&&& 5& 6&&& 52 5&&& 5" !&&& 61 3&&& 6" 2&&& "2 15&& "# 1&&& !& 1&8&& !5 8&&& 5& 5#&& 5" 38&& !& 1&8&& !575 "8&& 5& 5#&& 52 52&& 5675 38&& 52 52&& 5"75 3"&& 62 28&& 6! 25&& "" 13&& "8 12&&

(ig *